Isotopically-labeled benzofuran compounds as imaging agents for amyloidogenic proteins

ABSTRACT

Benzofuran compounds which contain at least one detectable label selected from the group consisting of  131 I,  123 I,  124 I,  76 Br,  75 Br,  18 F,  19 F,  11 C,  13 C,  14 C and  3 H are provided as amyloid imaging agents for detecting brain amyloid deposits as well as other amyloidogenic peptides associated with systemic or localized amyloidosis. Additionally, the compounds are useful for determining if patients, presenting with clinically confusing cases of dementia or presenting with mild cognitive impairment, have Alzheimer&#39;s disease. The compounds are additionally useful as surrogate markers for monitoring the efficacy of anti-amyloidosis therapies.

FIELD OF THE INVENTION

The present invention relates generally to the field ofisotopically-labeled benzofuran compounds which are substrates foramyloidogenic proteins, for example, Aβ1-42, an amyloid protein, braindeposits of which are associated with Alzheimer's disease.

BACKGROUND OF THE INVENTION I. Brain Amyloidosis

Alzheimer's Disease (“AD”) is a neurodegenerative illness characterizedby memory loss and other cognitive deficits. McKhann et al., Neurology34: 939 (1984). It is the most common cause of dementia in the UnitedStates. AD can strike persons as young as 40-50 years of age, yet,because the presence of the disease is difficult to determine withoutdangerous brain biopsy, the time of onset is unknown. The prevalence ofAD increases with age, with estimates of the affected populationreaching as high as 40-50% by ages 85-90. Evans et al., JAMA 262: 2551(1989); Katzman, Neurology 43: 13 (1993).

Studies suggest that amyloid deposition in the brain is an early,causative event in the pathogenesis of Alzheimer's disease (AD).Progression of amyloid deposition results in the formation of neuriticplaques and neurofibrillary tangles in regions of the brain that areinvolved with learning and memory. A typical Alzheimer's neuritic plaquecomprises dystrophic neurites surrounding a core of amyloid material.The principal component of the amyloid core is a protein calledamyloid-beta (Aβ).

In practice, AD is definitively diagnosed through examination of braintissue, usually at autopsy. Khachaturian, Arch. Neurol. 42: 1097 (1985);McKhann et al., Neurology 34: 939 (1984). Neuropathologically, thisdisease is characterized by the presence of neuritic plaques (NP),neurofibrillary tangles (NFT), and neuronal loss, along with a varietyof other findings. Mann, Mech. Ageing Dev. 31: 213 (1985). Post-mortemslices of brain tissue of victims of Alzheimer's disease exhibit thepresence of amyloid in the form of proteinaceous extracellular cores ofthe neuritic plaques that are characteristic of AD.

The amyloid cores of these neuritic plaques are composed of a proteincalled the β-amyloid (Aβ) that is arranged in a predominatelybeta-pleated sheet configuration. Mori et al., Journal of BiologicalChemistry 267: 17082 (1992); Kirschner et al., PNAS 83: 503 (1986).Neuritic plaques are an early and invariant aspect of the disease. Mannet al., J. Neurol. Sci. 89: 169; Mann, Mech. Ageing Dev. 31: 213 (1985);Terry et al., J. Neuropathol. Exp. Neurol 46: 262 (1987).

The initial deposition of Aβ probably occurs long before clinicalsymptoms are noticeable. The currently recommended “minimum microscopiccriteria” for the diagnosis of AD is based on the number of neuriticplaques found in brain. Khachaturian, Arch. Neurol. (1985), supra.Assessment of neuritic plaque counts must be delayed until after death,however.

Amyloid-containing neuritic plaques are a prominent feature of selectiveareas of the brain in AD as well as Down's Syndrome and in personshomozygous for the apolipoprotein E4 allele who are very likely todevelop AD. Corder et al., Science 261: 921 (1993); Divry, P., J.Neurol. Psych. 27: 643-657 (1927); Wisniewski et al., in Zimmerman, H.M. (ed.): PROGRESS IN NEUROPATHOLOGY (Grune and Stratton, N.Y. 1973) pp.1-26.

Brain amyloid is readily demonstrated by staining brain sections withthioflavin S or Congo red. Puchtler et al., J. Histochem. Cytochem. 10:35 (1962). Congo red-stained amyloid is characterized by a dichroicappearance, exhibiting a yellow-green polarization color. The dichroicbinding is the result of the beta-pleated sheet structure of the amyloidproteins. Glenner, G. N. Eng. J. Med. 302: 1283 (1980). A detaileddiscussion of the biochemistry and histochemistry of amyloid can befound in Glenner, N. Eng. J. Med., 302: 1333 (1980).

Thus far, diagnosis of AD has been achieved mostly through clinicalcriteria evaluation, brain biopsies and post-mortem tissue studies.Research efforts to develop methods for diagnosing Alzheimer's diseasein vivo include genetic testing, immunoassay methods, and imagingtechniques.

Evidence that abnormalities in Aβ metabolism are necessary andsufficient for the development of AD is based on the discovery of pointmutations in the Aβ precursor protein in several rare families with anautosomal dominant form of AD. Hardy, Nature Genetics 1: 233 (1992);Hardy et al., Science 256: 184 (1992). These mutations occur near the N-and C-terminal cleavage points necessary for the generation of Aβ fromits precursor protein. St. George-Hyslop et al., Science 235: 885(1987); Kang et al., Nature 325: 733 (1987); Potter WO 92/17152. Geneticanalysis of a large number of AD families has demonstrated, however,that AD is genetically heterogeneous. St. George-Hyslop et al., Nature347: 194 (1990). Linkage to chromosome 21 markers is shown in only somefamilies with early-onset AD and in no families with late-onset AD. Morerecently a gene on chromosome 14 whose product is predicted to containmultiple transmembrane domains and resembles an integral membraneprotein has been identified by Sherrington et al., Nature 375: 754-760(1995). This gene may account for up to 70% of early-onset autosomaldominant AD. Preliminary data suggests that this chromosome 14 mutationcauses an increase in the production of Aβ. Scheuner et al., Soc.Neurosci. Abstr. 21: 1500 (1995). A mutation on a very similar gene hasbeen identified on chromosome 1 in Volga German kindreds withearly-onset AD. Levy-Lahad et al., Science 269: 973-977 (1995).

Screening for apolipoprotein E genotype has been suggested as an aid inthe diagnosis of AD. Scott, Nature 366: 502 (1993); Roses, Ann. Neurol.38: 6-14 (1995). Difficulties arise with this technology, however,because the apolipoprotein E4 allele is only a risk factor for AD, not adisease marker. It is absent in many AD patients and present in manynon-demented elderly people. Bird, Ann. Neurol. 38: 2-4 (1995).

Immunoassay methods have been developed for detecting the presence ofneurochemical markers in AD patients and to detect an AD related amyloidprotein in cerebral spinal fluid. Warner, Anal. Chem. 59: 1203A (1987);World Patent No. 92/17152 b_(y) Potter; Glenner et al., U.S. Pat. No.4,666,829. These methods for diagnosing AD have not been proven todetect AD in all patients, particularly at early stages of the diseaseand are relatively invasive, requiring a spinal tap. Also, attempts havebeen made to develop monoclonal antibodies as probes for imaging of Aβ.Majocha et al., J. Nucl. Med., 33: 2184 (1992); Majocha et al., WO89/06242 and Majocha et al., U.S. Pat. No. 5,231,000. The majordisadvantage of antibody probes is the difficulty in getting these largemolecules across the blood-brain barrier. Using antibodies for in vivodiagnosis of AD would require marked abnormalities in the blood-brainbarrier in order to gain access into the brain. There is no convincingfunctional evidence that abnormalities in the blood-brain barrierreliably exist in AD. Kalaria, Cerebrovascular & Brain MetabolismReviews 4: 226 (1992).

Radiolabeled Aβ peptide has been used to label diffuse, compact andneuritic type plaques in sections of AD brain. See Maggio et al., WO93/04194. However, these peptides share all of the disadvantages ofantibodies. Specifically, peptides do not normally cross the blood-brainbarrier in amounts necessary for imaging and because these probes reactwith diffuse plaques, they may not be specific for AD.

Neuritic plaques and neurofibrillary tangles are the two mostcharacteristic pathological hallmarks of AD. Klunk and Abraham,Psychiatric Development, 6:121-152 (1988). Plaques occur earliest inneocortex where they are relatively evenly distributed. Thal et al.,Neurology 58:1791-1800 (2002). Tangles appear first in limbic areas suchas the transentorhinal cortex and progress in a predictable topographicpattern to the neocortex. Braak and Braak, Acta Neuropathologica82:239-259 (1991). Arnold et al. mapped the distribution of NFT andneuritic plaques in the brains of patients with AD. Arnold et al.,Cereb. Cortex 1:103-116 (1991). Compared to NFT, neuritic plaques were,in general, more evenly distributed throughout the cortex, with theexceptions of notably fewer neuritic plaques in limbic periallocortexand allocortex (the areas with greatest NFT density). By thioflavin-Sstaining, temporal and occipital lobes had the highest neuritic plaquedensities, limbic and frontal lobes had the lowest, and parietal lobewas intermediate. Arriagada et al., Neurology 42:1681-1688 (1992).Arriagada et al studied the topographic distribution of AD-typepathologic changes in the brains of presumed nondemented elderlyindividuals. Their observations suggest that most individuals over theage of 55 have at least a few NFT and plaques. Immunohistochemicallydefined subtypes of SP had distinct patterns of distribution withAβ-immunoreactive plaques present in neocortical areas much greater thanlimbic areas and Alz-50 immunoreactive plaques being infrequent andlimited to those areas that contain Alz-50-positive neurons and NFT.These patterns suggested a commonality in the pathologic processes thatlead to NFT and SP in both aging and AD.

There remains debate as to whether plaques and tangles are byproducts ofthe neurodegenerative process found in AD or whether they are the causeof neuronal cell death. Ross, Current Opinion in Neurobiol. 96:644-650(1996); Terry, J. of Neuropath. & Exp. Neurol. 55:1023-1025 (1996);Terry, J Neural Transmission-Suppl. 53:141-145 (1998). Evidence is clearthat neocortical and hippocampal synapse loss correlates well withpre-morbid cognitive status. Some researchers suggest that disruption ofmicrotubule structure and function, caused by the hyperphosphorylationof the microtubule-associated protein, tau, plays the key etiologic rolein synapse loss in particular and AD in general. Terry, J. of Neuropath.& Exp. Neurol. 55:1023-1025 (1996); Terry, J of NeuralTransmission-Suppl. 53:141-145 (1998). Oxidative damage and membranebreakdown have been proposed to play important roles in AD. Perry, FreeRadical Biology & Medicine 28:831-834 (2000); Pettegrew et al., Annalsof the New York Academy of Sciences 826:282-306 (1997). Vascular factorsincluding subtle, chronic cerebral hypoperfusion also have beenimplicated in the pathogenesis of AD. De la Torre, Annals of the NewYork Academy of Sciences 903:424-436 (2000); Di Iorio et al., Aging(Milano) 11:345-352 (1999). While all of these factors are likely toplay some role in the pathogenesis of AD, increasing evidence points toabnormalities in the processing of the amyloid-beta (Aβ) peptide, a 4 kDpeptide that aggregates into a fibrillar, β-pleated sheet structure.Glenner and Wong, Biochemical & Biophysical Research Communications120:885-890 (1984). Aβ has been proposed to play an important role inthe pathogenesis of AD for several reasons: 1) Aβ deposits are theearliest neuropathological markers of AD in Down's Syndrome, and canprecede NFT formation by several decades Mann et al., Neurodegeneration1:201-215 (1992); Naslund, et al., JAMA 283:1571-1577 (2000). 2)β-amyloidosis is relatively specific to AD and closely relateddisorders; Selkoe, Trends in Neurosciences 16:403-409 (1993); 3) Aβ istoxic to cultured neurons, Yankner Neurobiol. Aging 13:615-616 (1992);Mattson et al., J. Neuroscience 12:376-389 (1992); Shearman et al.,Proc. Natl. Acad. Sci. USA 91:1470-1474 (1994), a toxicity that appearsto be dependent on β-sheet secondary structure and aggregation into atleast oligomers. Lambert et al. Proc. Natl. Acad. Sci. USA 95:6448-6453(1989) ; Pike et al., J. Neuroscience 13:1676-1687 (1993) ; Simmons etal., Molecular Pharmacology 45:373-379 (1994). Although Aβ surely existsin an equilibrium distributed across monomeric, oligomeric andfibrillar/plaque fractions, the oligomeric form of Aβ has been stronglyimplicated as the key neurotoxic component. Selkoe, Alzheimer disease,edited by R. D. Terry, et al, pp. 293-310 Lippincott Williams andWilkins, Philadelphia (1999); Selkoe, Science 298, 789-91 (2002).Recognition of the toxic effects of oligomeric Aβ has formed a basis forcompromise for some opponents of the “amyloid cascade hypothesis” of AD.Terry, Ann. Neurol. 49:684 (2001). Perhaps the strongest evidence for arole of Aβ in the pathogenesis of AD comes from the finding of mutationsin the amyloid precursor protein (APP) gene which lead to some forms ofearly onset familial AD. Goate et al., Nature 349:704-706 (1991). Inaddition, all familial forms of autosomal dominant AD have in common anelevated level of the more rapidly aggregating 42 amino acid form of Aβ.Younkin Rinsho Shinkeigaku—Clinical Neurology 37:1099 (1997). Incontrast, no mutation in the tau protein has been shown to cause AD.Instead mutations in tau (chromosome 17) are linked to frontotemporaldementia with Parkinsonism. Goedert et al., Neuron 21:955-958 (1998).Recent evidence has shown a good correlation between the levels of Aβ inbrain and cognitive decline in AD and the deposition of amyloid appearsto be a very early, perhaps the first, event in the pathogenesis of AD,preceding any cognitive impairment. Naslund, et al., JAMA 283:1571-1577(2000). The presence of amyloid deposits may modulate a number ofbiochemical pathways that result in the deposition of still otherproteins, the activation of astroglia and microglia, and eventuallyneuronal cell death and consequent cognitive dysfunction.

II. Localized and Systemic Amyloidosis

Amyloidosis is a slowly progressive condition, which can lead tosignificant morbidity and death. A diverse group of disease processesfall under the “amyloidosis” rubric, which is characterized byextracellular tissue deposits, in one or many organs, of variousinsoluble fibrillar proteins, generically termed “amyloid,” in amountssufficient to impair normal function.

Amyloid deposits are extracellular and not metabolized or cleared by thebody. Amyloid may be distinguished grossly by a starch-like stainingreaction with iodine; hence the name amyloid. Microscopically, amyloidis differentiated by its extracellular distribution, by its tinctorialand optical properties when stained with Congo red, and by its proteinfibril structure. Thus, under light microscopy, amyloid is ahomogeneous, highly refractile substance with an affinity for Congo reddye, both in fixed tissues and in vivo. Under electron microscopy,amyloid consists of 100 A° (10 nm), linear nonbranching fibrils; underx-ray diffraction, it has a cross-beta pattern.

The diseases associated with amyloidosis are all typified by anaccumulation amyloid deposits. The amyloid deposits are characterized bythe presence of one or more amyloidogenic proteins, which are derivedfrom precursor proteins that either have an abnormal structure or areabnormally increased in the serum.

The cause of amyloid production and its deposition in tissues isunknown. In the different biochemical types of amyloidosis, etiologicmechanisms may vary. In secondary amyloidosis, for example, a defect inthe metabolism of the precursor protein (the acute-phase reactant: serumamyloid A) may exist, whereas in hereditary amyloidosis a geneticallyvariant protein appears to be present. In primary amyloidosis, amonoclonal population of marrow cells produces fragments of or wholelight chains that may be processed abnormally to form amyloid.

Three major types of amyloid and several less common forms have beendefined biochemically. The first type, which has an N-terminal sequencethat is homologous to a portion of the variable region of animmunoglobulin light chain, is called AL and occurs in primaryamyloidosis and in amyloidosis associated with multiple myeloma. Thesecond type has a unique N-terminal sequence of a nonimmunoglobulinprotein called AA protein and occurs in patients with secondaryamyloidosis. The third type, which is associated with familial amyloidpolyneuropathy, is usually a transthyretin (prealbumin) molecule thathas a single amino acid substitution. Other hereditary amyloids havebeen found to consist of mutant gelsolin in some families, mutantapolipoprotein A-I in several others, and other mutant proteins inhereditary cerebral artery amyloid. In the amyloid associated withchronic hemodialysis, 2-microglobulin has constituted amyloid protein.Amyloid associated with aging in skin and with endocrine organs mayrepresent other biochemical forms of amyloidosis. The amyloid found inthe histopathologic lesions of Alzheimer's disease consists of proteins.Chemical analyses relating to various forms of amyloidosis have led to amore refined classification. A unique protein, a pentraxin called AP (orserum AP), is universally associated with all forms of amyloid and formsthe basis of a diagnostic test.

Three major systemic clinical forms are recognized currently.Amyloidosis is classified as primary or idiopathic (AL form) when thereis no associated disease, and secondary, acquired, or reactive (AA form)when associated with chronic diseases, either infectious (tuberculosis,bronchiectasis, osteomyelitis, leprosy) or inflammatory (rheumatoidarthritis, granulomatous ileitis). Amyloid also is associated withmultiple myeloma (AL), Hodgkin's disease (AA), other tumors, andfamilial Mediterranean fever (AA). Amyloidosis may accompany aging. Thethird major type appears in familial forms unassociated with otherdisease, often with distinctive types of neuropathy, nephropathy, andcardiopathy.

In primary (AL) amyloidosis, the heart, lung, skin, tongue, thyroidgland, and intestinal tract may be involved. Localized amyloid “tumors”may be found in the respiratory tract or other sites. Parenchymal organs(liver, spleen, kidney) and the vascular system, especially the heart,are involved frequently.

Secondary (AA) amyloidosis shows a predilection for the spleen, liver,kidney, adrenals, and lymph nodes. No organ system is spared, however,and vascular involvement may be widespread, though clinicallysignificant involvement of the heart is rare. The liver and spleen oftenare enlarged, firm, and rubbery. The kidneys usually are enlarged.Sections of the spleen have large, translucent, waxy areas where thenormal malpighian bodies are replaced by pale amyloid, producing thesago spleen.

Hereditary amyloidosis is characterized by a peripheral sensory andmotor neuropathy, often autonomic neuropathy, and cardiovascular andrenal amyloid. Carpal tunnel syndrome and vitreous abnormalities mayoccur.

Amyloid associated with certain malignancies (e.g., multiple myeloma)has the same distribution as idiopathic (AL) amyloid; with othermalignancies (e.g., medullary carcinoma of the thyroid gland) it mayoccur only locally in association with the tumor or in metastases.Amyloid frequently is found in the pancreas of individuals withadult-onset diabetes mellitus.

While amyloidosis may be suspected on the basis of specific clinicalsymptoms and signs, it can be definitively diagnosed only by biopsy.Currently, subcutaneous abdominal fat pad aspiration and biopsy ofrectal mucosa are the best screening tests. Other useful biopsy sitesare gingiva, skin, nerve, kidney, and liver. Tissue sections should bestained with Congo red dye and observed with a polarizing microscope forthe characteristic green birefringence of amyloid. Isotopically labeledserum AP has been used in a scintigraphic test to confirm the diagnosisof amyloidosis. Better diagnostic methodologies need to be developed inorder to provide early diagnosis thereby permitting effective treatment.

There is some speculation of a connection between inhibition of amyloiddeposits and diabetes therapy, see, e.g., WO 02/16333. Imaging of thepancreas for diagnosing diabetes is one suitable methodology fordefinitively measuring the levels of amyloid in the pancreas, acorrelation of which appears to be indicative of a diagnosis ofdiabetes.

III. Surrogate Markers

AD is believed to afflict some 4 million Americans and perhaps 20-30million people worldwide. AD is recognized as a major public healthproblem in developed nations. Several therapeutic targets have emergedfrom the ongoing elucidation of the molecular basis of AD. For example,four cholinesterase inhibitors have been approved for the symptomatictreatment of patients with AD—tacrine (Cognex, Warner-Lambert, MorrisPlains, N.J.); donepezil (Aricept, Eisai, Inc., Teaneck, N.J., andPfizer, Inc., New York, N.Y.); rivagstigmine (Exelon, Novartis, Basel,Switzerland); and galantamine (Reminyl, Janssen, Titusville, N.J.).Potential new AD therapies that are currently being developed involveimmunotherapy, secretase inhibitors or anti-inflammatory drugs. However,to date, there are no available drugs proven to modify the course ofcognitive decline.

A major hurdle to developing anti-amyloid therapies is exemplified bythe following quote from (Hock, C. et al., 2003, Neuron, 38:547-554),directed to use of immunotherapy as an anti-amyloid therapy: “[w]e donot know whether brain Aβ-amyloid load was reduced in our studypatients; in vivo imaging techniques will be required to answer thisquestion.” The ability to quantify amyloid load before treatment andthen follow the effects of treatment is critical to the efficientdevelopment of this class of drugs.

IV. Diagnosing Prodromal Forms of Amyloidosis

A condition closely related to Alzheimer's Disease (AD) is characterizedby either isolated memory impairment or impairment in several cognitivedomains, but not of sufficient severity to meet diagnostic criteria forAlzheimer's disease. This condition has been termed mild cognitiveimpairment (MCI) and may represent a prodromal phase of AD. Mildcognitive impairment is defined as an intermediate or transitional statefrom a normal cognitive state to dementia. Subjects with a mildcognitive impairment typically have a memory impairment beyond thatexpected for age and education yet are not demented.

There is some indication that patients diagnosed with mild cognitiveimpairment will progress to AD. There is also indications that mildcognitive impairment may represent a complex heterogeneous condition andthat some patients with mild cognitive impairment will not develop AD orother dementing disorders.

There have been volumes of interest in discerning the boundary ofdementia to AD. Most of the interest deals with a boundary ortransitional state between normal aging and dementia, or morespecifically, Alzheimer disease (AD). Reviews of several studies haveindicated that these individuals are at an increased risk for developingAD ranging from 1% to 25% per year. The variability in these rateslikely reflects differing diagnostic criteria, measurement instruments,and small sample sizes. See Dawe et al., Int'l J. Geriatr. Psychiatry 7:473 (1992).

Patients diagnosed with an MCI are also becoming of interest fortreatment trials. The Alzheimer's Disease Cooperative Study, which is aNational Institute on Aging consortium of Alzheimer's Disease researchgroups, is embarking on a multicenter trial of agents intended to alterthe progression of patients with MCI to AD. See Grundman et al.,Neurology, 1996, A403.

Questions can be raised as to the diagnostic criteria for MCI. Someinvestigators believe that virtually all these patients with milddisease have AD neuropathologically, and, therefore, this may not be auseful distinction. See Morris et al., Neurology 41: 469 (1991). Othersnote that, while many of these patients progress to AD, not all do and,consequently, that the distinction is an important one. See Grundman,ibid; Petersen et al., JAMA 273: 1274 (1995); Petersen et al., Ann N YAcad. Sci. 802: 58 (1996).

V. Substrates for Amyloidogenic Proteins-Radiolabeled Benzofurans

Potential substrates for amyloidogenic proteins have been postulated andrange from dye substances, such as Congo Red and derivatives ofChrysamine G (see, e.g., U.S. Pat. No. 6,168,776) to sequence specificpeptides that have been labeled for the purpose of imaging insolubleA-beta. These peptides includes the labeled A-beta peptide itself,putrescine-gadolinium-A-beta peptide, radiolabeled A-beta,[¹¹¹In]A-beta, [¹²⁵I]A-beta, A-beta labeled with gamma emittingradioisotopes, A-beta-DTPA derivatives, radiolabeled putrescine,KVLFF-based ligands and derivatives thereof (see, e.g., InternationalPub. No. WO93/04194 and U.S. Pat. No. 6,331,440).

A few non-radiolabeled benzofurans purported for use in diagnosingAlzheimer's disease were described in U.S. Pat. No. 6,696,039. However,none of the benzofuran compounds disclosed were radiolabeled and nonewere disclosed to be effective for imaging Alzheimer's disease in aliving patient.

Although some labeled benzofurans are known, none have been used indiagnosing Alzheimer's disease. See Aitken et al., Journal of theChemical Society, Perkin Transactions 1: Organic and Bio-OrganicChemistry (1998), (23), 3937-3942 disclosing a deuterated benzofuranderivative; Givens et al. disclose 2-phenyl-benzofuran-d in Journal ofthe American Chemical Society (1993) 115(14), 6001-12 and Davies et al.disclose ¹⁴C labeled 2-phenyl-benzofuran in Journal of the ChemicalSociety (1959) 3544-7.

A need therefore exists for isotopically-labeled benzofurans having thatare capable of crossing the blood brain barrier and binding to insolubleamyloid deposits for imaging in diagnosing Alzheimer's disease.

SUMMARY OF THE INVENTION

The present invention satisfies this need and others by providing, inone embodiment, an amyloid binding compound of Formula (I) or apharmaceutically acceptable salt thereof:

In formula (I), Y is H, NO₂, —NR″₃ ⁺, F, Cl, Br, I, —O—(CR″₂)_(n)—X, or—(CR″₂)_(n)—X, wherein X is F, Cl, Br or I. Variable n is an integerthat is selected from 1-5.

R′ is H or a lower alkyl group. Too, R″ is H or a lower alkyl group.

R₃-R₁₃ are independently selected from the group consisting of H, F, Cl,Br, I, C₁-C₅ alkyl, (CH₂)₁₋₃—OR₁₁, CF₃, —(CH₂)₁₋₃—X, —O—(CH₂)₁₋₃—X, CN,—CO—R₁₁, —N(R₁₁)₂, —NR″₃ ⁺, —NO₂, —CO—N(R₁₁)₂, —O—(CO)—R₁₁, OR₁₁, SR₁₁,COOR₁₁, R_(ph), —CR₁₁═CR₁₁—R_(ph) and —C(R₁₁)₂—C(R₁₁)₂—R_(ph). Asmentioned above, X is F, Cl, Br or I. R_(ph) is phenyl optionallysubstituted with one or more substituents selected from the groupconsisting of F, Cl, Br, I, C₁-C₅ alkyl, (CH₂)₁₋₃—OR₁₁, CF₃,—(CH₂)₁₋₃—X, —O—(CH₂)₁₋₃—X, CN, —CO—R₁₁, —N(R₁₁)₂, —CO—N(R₁₁)₂,—O—(CO)—R₁₁, OR₁₁, SR₁₁, and COOR₁₁, wherein each R₁₁ is independently Hor C₁-C₅ alkyl,

Additionally, substituent Y or R³-R¹⁰ comprises at least one detectablelabel selected from the group consisting of ¹³¹I, ¹²³I, ¹²⁴I, ¹²⁵I,⁷⁶Br, ⁷⁵Br, ¹⁸F, ¹⁹F, ¹¹C, ¹³C, ¹⁴C and ³H.

In another embodiment, there is provided a pharmaceutical compositioncomprising an effective amount of an amyloid binding compound accordingformula (I) as described above and a pharmaceutically acceptablecarrier.

Yet another embodiment is a method for detecting amyloid deposit(s) invivo. The method comprises (i) administering to a mammal an effectiveamount of an amyloid binding compound according formula (I), wherein thecompound would bind any amyloid deposit(s) in the mammal; and (ii)detecting binding of the compound to amyloid deposit(s) in the mammal.

Additionally, alone or in combination with any other embodiment hereindescribed, the invention encompasses the use of a compound according toformula (I), as herein defined, for detecting amyloid deposits in vivoin a mammal. In a related embodiment, the invention further provides forthe use of a compound of formula (I) in the preparation of a medicamentfor use in the detection of amyloid deposits in vivo in such a subject.

Still another embodiment is a method for detecting amyloid deposit(s) invitro. The method comprises (i) contacting a bodily tissue with aneffective amount of an amyloid binding compound according to formula(I), wherein the compound would bind any amyloid deposit(s) in thetissue; and (ii) detecting binding of the compound to amyloid deposit(s)in the tissue.

Alone or in combination with any other embodiment herein described,another embodiment is the use of a compound according to formula (I), asherein defined, for detecting amyloid deposits in vitro. In anotherembodiment, the invention provides for the use of a compound of formula(I) in the preparation of a medicament for use in the detection ofamyloid deposits in vitro.

An additional embodiment is a method for distinguishing an Alzheimer'sdiseased brain from a normal brain. The method comprises (i) obtainingtissues from (i) the cerebellum and (ii) another area of the same brain,of a normal mammal and of a mammal suspected of having Alzheimer'sdisease;

(ii) contacting the tissues with an amyloid binding compound accordingto claim 1;

(iii) quantifying the amyloid bound to the compound;

(iv) calculating the ratio of (a) the amount of amyloid in the area ofthe brain other than the cerebellum to (b) the amount of amyloid in thecerebellum; and

(v) comparing the ratio for a normal mammal with the ratio for a mammalsuspected of having Alzheimer's disease.

In another embodiment, alone or in combination with any other embodimentherein described, the invention provides the use of a compound accordingto formula (I), as herein defined, for distinguishing an Alzheimer'sdiseased brain from a normal brain. Another embodiment is the use of aformula (I) compound in the preparation of a medicament for use indistinguishing an Alzheimer's diseased brain from a normal brain.

Yet another embodiment is a method of detecting amyloid deposits inbiopsy or post-mortem human or animal tissue. The method comprises thesteps of (a) incubating formalin-fixed or fresh-frozen tissue with asolution of an amyloid binding compound of Formula (I) or apharmaceutically acceptable salt thereof to form a labeled deposit and(b) detecting the labeled deposit.

In another embodiment, alone or in combination with any other embodimentherein described, the invention provides the use of a compound accordingto formula (I), as herein defined, for detecting amyloid deposits inbiopsy or post-mortem human or animal tissue. Another embodiment is theuse of a formula (I) compound in the preparation of a medicament for usein detecting amyloid deposits in biopsy or post-mortem human or animaltissue.

In still another embodiment, there is provided a method of quantifyingthe amount of amyloid in biopsy or post-mortem tissue. The methodcomprises the steps of:

a) incubating a radiolabeled derivative of an amyloid binding compoundof formula (I) or a pharmaceutically acceptable salt thereof with ahomogenate of biopsy or post-mortem tissue, wherein at least one of thesubstituents in the compound is labeled with a radiolabel selected fromthe group consisting of ¹²⁵I, ³H, and a carbon-containing substituent,wherein at least one carbon is ¹⁴C;

b) separating the tissue-bound from the tissue-unbound radiolabeledderivative of a compound of formula (I),

c) quantifying the tissue-bound radiolabeled derivative of a compound offormula (I), and

d) converting the units of tissue-bound radiolabeled derivative of acompound of formula (I) to units of micrograms of amyloid per 100 mg oftissue by comparison with a standard.

In another embodiment, alone or in combination with any other embodimentherein described, the invention provides the use of a compound accordingto formula (I), as herein defined, for quantifying the amount of amyloidin biopsy or post-mortem tissue. Another embodiment is the use of aformula (I) compound in the preparation of a medicament for use inquantifying the amount of amyloid in biopsy or post-mortem tissue.

In another embodiment, there is provided a method of selectively bindingan amyloid binding compound of Formula (I) or a pharmaceuticallyacceptable salt thereof to amyloid plaques but not neurofibrillarytangles in brain tissue which contains both. The method comprisescontacting the amyloid plaques in in vitro binding or staining assayswith a compound of Formula (I) at a concentration below about 10 nM.

In yet another embodiment, alone or in combination with any otherembodiment herein described, the invention provides the use of acompound according to formula (I), as herein defined, for selectivelybinding an amyloid binding compound of Formula (I) or a pharmaceuticallyacceptable salt thereof to amyloid plaques but not neurofibrillarytangles in brain tissue which contains both. Another embodiment is theuse of a formula (I) compound in the preparation of a medicament for usein selectively binding an amyloid binding compound of Formula (I) or apharmaceutically acceptable salt thereof to amyloid plaques but notneurofibrillary tangles in brain tissue which contains both.

In yet another embodiment, there is provided a method of selectivelybinding in vivo an amyloid binding compound of Formula (I) or apharmaceutically acceptable salt thereof to amyloid plaques but not toneurofibrillary tangles in brain tissue which contains both. The methodcomprises administering an effective amount of a compound of Formula (I)or a pharmaceutically acceptable salt thereof such that the bloodconcentration of the administered compound remains below about 10 nM invivo.

Still another embodiment is an in vivo or in vitro method for detectingin a subject at least one amyloid deposit comprising at least oneamyloidogenic protein. The method comprises the steps of:

(a) administering to a subject suffering from a disease associated withamyloidosis, a detectable quantity of a pharmaceutical compositioncomprising at least one amyloid binding compound of formula I or apharmaceutically acceptable salt thereof and a pharmaceuticallyacceptable carrier, and

(b) detecting the binding of the compound to an amyloid depositcomprising at least one amyloidogenic protein.

In yet another embodiment, alone or in combination with any otherembodiment herein described, the invention provides the use of acompound according to formula (I), as herein defined, for the in vitroor in vivo detection in a subject of at least one amyloid depositcomprising at least one amyloidogenic protein. Another embodiment is theuse of a formula (I) compound in the preparation of a medicament for thein vitro or in vivo detection in a subject of at least one amyloiddeposit comprising at least one amyloidogenic protein.

In still another embodiment, there is provided a method of identifying apatient as prodromal to a disease associated with amyloid depositioncomprising:

(A) administering to the patient, who is presenting with signs ofclinical dementia or clinical signs of a mild cognitive impairment, anamyloid binding compound of formula (I) or a pharmaceutically acceptablesalt thereof; then

(B) imaging said patient to obtain data; and

(C) analyzing said data to ascertain amyloid levels in said patient withreference to a nonnative level, thereby identifying said patient asprodromal to a disease associated with amyloid deposition.

In still another embodiment, alone or in combination with any otherembodiment herein described, the invention provides the use of acompound according to formula (I), as herein defined, for identifying apatient as prodromal to a disease associated with amyloid deposition.Another embodiment is the use of a formula (I) compound in thepreparation of a medicament for identifying a patient as prodromal to adisease associated with amyloid deposition.

In another embodiment, there is provided a method of determining theefficacy of therapy in the treatment of amyloidosis. The methodcomprises

(A) administering to a patient in need thereof an effective amount of anamyloid binding compound of formula (I) or a pharmaceutically acceptablesalt thereof;

(B) imaging said patient; then

(C) administering to said patient in need thereof at least oneanti-amyloid agent;

(D) subsequently administering to said patient in need thereof aneffective amount of a compound of formula (I);

(E) imaging said patient; and

(F) comparing levels of amyloid deposition in said patient beforetreatment with said at least one anti-amyloid agent to levels of amyloiddeposition in said patient after treatment with said at least oneanti-amyloid agent.

In still another embodiment, alone or in combination with any otherembodiment herein described, the invention provides the use of acompound according to formula (I), as herein defined, for determiningthe efficacy of therapy in the treatment of amyloidosis. Anotherembodiment is the use of a formula (I) compound in the preparation of amedicament for determining the efficacy of therapy in the treatment ofamyloidosis.

DETAILED DESCRIPTION OF THE INVENTION

The present invention exploits the ability of isotopically-labeledbenzofuran derivatives to cross the blood brain barrier in vivo and bindto amyloidogenic proteins.

One example of such binding is the ability of the benzofuran compoundsto bind to Aβ deposited in neuritic (but not diffuse) plaques, to Aβdeposited in cerebrovascular amyloid, and to the amyloid consisting ofthe protein deposited in NFT.

Characterization of Specific Binding to Aβ Synthetic Peptide: Affinity,Kinetics, Maximum Binding

The characteristics of benzofuran derivative binding were analyzed usingsynthetic Aβ(1-40) and 2-(4′-[³H]methylamino-phenyl)-benzothiazole([³H]-BTA-1) in phosphate-buffered saline (pH 7.4) as previouslydescribed. Klunk et al., Life Sci. 69:1471 (2001); Mathis et al.,Bioorg. Med. Chem. Lett., 12:295 (2002).

Amino Acid Sequence for Aβ(1-40) is as follows:

1 2 3 4 5 6 7 8 9 10 Asp Ala Glu Phe Arg His Asp Ser Gly Tyr 11 12 13 1415 16 17 18 19 20 Glu Val His His Gln Lys Leu Val Phe Phe 21 22 23 24 2526 27 28 29 30 Ala Glu Asp Val Gly Ser Asn Lys Gly Ala 31 32 33 34 35 3637 38 39 40 Ile Ile Gly Leu Met Val Gly Gly Val Val

DEFINITIONS

“Alkyl” refers to a saturated straight or branched chain hydrocarbonradical. Examples include without limitation methyl, ethyl, propyl,iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl and n-hexyl.

“Alkenyl” refers to an unsaturated straight or branched chainhydrocarbon radical comprising at least one carbon to carbon doublebond. Examples include without limitation ethenyl, propenyl,iso-propenyl, butenyl, iso-butenyl, tert-butenyl, n-pentenyl andn-hexenyl.

“Alkynyl” refers to an unsaturated straight or branched chainhydrocarbon radical comprising at least one carbon to carbon triplebond. Examples include without limitation ethynyl, propynyl,iso-propynyl, butynyl, iso-butynyl, tert-butynyl, pentynyl and hexynyl.

“Alkoxy” refers to an alkyl group bonded through an oxygen linkage.

“Lower” used in combination with alkyl, alkenyl, alkynyl or alkoxyrefers to C₁-C₈ moieties.

“Halo” refers to a fluoro, chloro, bromo or iodo radical.

“Radioactive halo” refers to a radioactive halo, i.e. radiofluoro,radiochloro, radiobromo or radioiodo.

“Effective amount” refers to the amount required to produce a desiredeffect. Examples of an “effective amount” include amounts that enabledetecting and imaging of amyloid deposit(s) in vivo or in vitro, thatyield acceptable toxicity and bioavailability levels for pharmaceuticaluse, and/or prevent cell degeneration and toxicity associated withfibril formation.

“Pharmaceutically acceptable carrier” refers to a pharmaceuticallyacceptable material, composition or vehicle, such as a liquid or solidfiller, diluent, excipient or solvent encapsulating material, involvedin carrying or transporting the subject compound from one organ, orportion of the body, to another organ or portion of the body. Eachcarrier is “acceptable” in the sense of being compatible with the otheringredients of the formulation and suitable for use with the patient.Examples of materials that can serve as a pharmaceutically acceptablecarrier include without limitation: (1) sugars, such as lactose, glucoseand sucrose; (2) starches, such as corn starch and potato starch; (3)cellulose and its derivatives, such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5)malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter andsuppository waxes; (9) oils, such as peanut oil, cottonseed oil,safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10)glycols, such as propylene glycol; (11) polyols, such as glycerin,sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyloleate and ethyl laurate; (13) agar; (14) buffering agents, such asmagnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16)pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19)ethyl alcohol; (20) pH buffered solutions; (21) polyesters,polycarbonates and/or polyanhydrides; and (22) other non-toxiccompatible substances employed in pharmaceutical formulations asidentified, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, 15^(th)ed. (Mack Publishing Co., 1975), at pages 1405-1412 and 1461-1487, andTHE NATIONAL FORMULARY XIV, 14^(th) ed. (American PharmaceuticalAssociation, 1975).

“Pharmaceutically acceptable salt” refers to, an acid or base salt ofthe inventive compound, which salt possesses the desired pharmacologicalactivity and is neither biologically nor otherwise undesirable. The saltcan be formed with acids that include without limitation acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfatebutyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloridehydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate and undecanoate. Examples of a base salt includewithout limitation ammonium salts, alkali metal salts such as sodium andpotassium salts, alkaline earth metal salts such as calcium andmagnesium salts, salts with organic bases such as dicyclohexylaminesalts, N-methyl-D-glucamine, and salts with amino acids such as arginineand lysine. In some embodiments, the basic nitrogen-containing groupscan be quarternized with agents including lower alkyl halides such asmethyl, ethyl, propyl and butyl chlorides, bromides and iodides; dialkylsulfates such as dimethyl, diethyl, dibutyl and diamyl sulfates; longchain halides such as decyl, lauryl, myristyl and stearyl chlorides,bromides and iodides; and aralkyl halides such as phenethyl bromides.

“Prodrug” refers to a derivative of the inventive compound thatundergoes biotransformation, such as metabolism, before exhibiting itspharmacological effect(s). The prodrug is formulated with theobjective(s) of improved chemical stability, improved patient acceptanceand compliance, improved bioavailability, prolonged duration of action,improved organ selectivity, improved formulation (e.g., increasedhydrosolubility), and/or decreased side effects (e.g., toxicity). Theprodrug can be readily prepared from the inventive compound usingconventional methods, such as that described in BURGER′S MEDICINALCHEMISTRY AND DRUG CHEMISTRY, 5^(th) ed., Vol. 1 (1995), pages 172-178and 949-982.

The term “parenteral” as used herein includes subcutaneous, intravenous,intraarterial, intramuscular, intraperitoneal, intrathecal,intraventricular, infrasternal, intracranial, and intraosseous injectionand infusion techniques.

“Animal” refers to a living organism having sensation and the power ofvoluntary movement, and which requires for its existence oxygen andorganic food. Examples include, without limitation, members of thehuman, equine, porcine, bovine, murine, canine and feline species. Inthe case of a human, an “animal” also may be referred to as a “patient.”

“Mammal” refers to a warm-blooded vertebrate animal.

A “subject” is a mammal, such as, for example, a human. A specificexample is a human suspected of having dementia.

“Treating” refers to:

(i) preventing a disease, disorder or condition from occurring in ananimal that may be predisposed to the disease, disorder and/or conditionbut has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and/or

(iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

The term “therapy” includes treating and/or preventing disease.

The term “treating” or “treatment” does not necessarily mean total cure.Any alleviation of any undesired symptom or pathological effect of thedisease to any extent or the slowing down of the progress of the diseasecan be considered treatment. Furthermore, treatment may include actswhich may worsen the patient's overall feeling of well being orappearance. For example, the administration of chemotherapy in cancerpatients which may leave the patients feeling “sicker” is stillconsidered treatment.

The term “preventing” refers to decreasing the probability that anorganism contracts or develops a disease associated with amyloiddeposition. For example, the term “preventing” refers to reducing thepercentage of individuals who develop the disease relative to a controlgroup that does not undergo administration of an anti-amyloid agent.

A “detectable quantity” means that the amount of the detectable compoundthat is administered is sufficient to enable detection of binding of thecompound to amyloid.

An “imaging effective quantity” means that the amount of the detectablecompound that is administered is sufficient to enable imaging of bindingof the compound to amyloid.

The term “in vivo imaging” refers to any method which permits thedetection of a labeled benzofuran compound as described herein.

The term “in vivo or in vitro method for detecting” refers to any methodwhich permits the detection of a labeled thioflavin derivative offormula (I).

The term “baseline” refers to the amount and distribution of a patient'samyloid deposition prior to initiation of the anti-amyloid therapy.

Unless the context clearly dictates otherwise, the definitions ofsingular terms may be extrapolated to apply to their plural counterpartsas they appear in the application; likewise, the definitions of pluralterms may be extrapolated to apply to their singular counterparts asthey appear in the application.

Amyloid Imaging Agents

The amyloid imaging agent of the present invention is any compound offormula (I) or a pharmaceutically acceptable salt thereof:

wherein R′, R₃-R₁₀, and Y are as defined above.

Compounds of formula (I), also referred to herein as “benzofurancompounds,” “benzofuran derivatives,” or “amyloid imaging agents,” haveeach of the following characteristics: (1) specific binding to syntheticAβ in vitro, (2) ability to cross a non-compromised blood brain barrierin vivo, (3) specific binding an amyloid deposit which comprises atleast one amyloidogenic protein, wherein the amyloidogenic protein isselected from the group consisting of AL, AH, ATTR, Aβ2M, AA, AApoAI,AApoAII, AGel, ALys, AFib, ACys, ABri, ADan, APrP, ACal, AlAPP, AANF,APro, AIns, AMed, AKer, A(tbn), and ALac, (4) bind to Aβdeposited inneuritic (but not diffuse) plaques, to Aβ deposited in cerebrovascularamyloid, and to the amyloid consisting of the protein deposited in NFTand (5) are also non-toxic at appropriate dosage levels and have asatisfactory duration of effect.

In one embodiment, optionally in combination with any other embodimentherein described, Y can be H, NO₂, —NR″₃ ⁺, F, Cl, Br, I, or—(CR″₂)_(n)—X.

In one embodiment, R₃-R₁₀ can be independently selected from the groupconsisting of H, F, Cl, Br, I, 1'N(R₁₁)₂, and OR₁₁. In combination withthis or any other embodiment herein described, R₈ and R₉ can beindependently OR₁₁.

In another embodiment optionally in combination with other embodimentsherein described, each of R₇ and R₁₀ can be H. In yet anotherembodiment, each of R₃, R₄, R₅, and R₆ can be H.

In still other embodiments of the invention, Y can be F, Cl, Br, I, or—NO₂. A specific example of Y is F.

In another embodiment, the amyloid binding compound of formula (I)provides for each of R₃, R₄, R₅, and R₆, R₇, and R₁₀ to be H, and R₈ andR₉ to be independently OR₁₁.

Another embodiment provides for Y to comprise at least one detectablelabel.

Illustrative compounds of formula (I) include but are not limited to:

Methods of Use

The inventive compounds may be used to determine the presence, locationand/or amount of one or more amyloid deposit(s) in an organ or bodyarea, including the brain, of an animal. Amyloid deposit(s) include,without limitation, deposit(s) of Aβ. In allowing the temporal sequenceof amyloid deposition to be followed, the inventive compound may furtherbe used to correlate amyloid deposition with the onset of clinicalsymptoms associated with a disease, disorder or condition. The inventivecompounds may ultimately be used to diagnose a disease, disorder orcondition characterized by amyloid deposition, such as AD, familial AD,Down's syndrome, amyloidosis, Type II diabetes mellitus, mild cognitiveimpairment and homozygotes for the apolipoprotein E4 allele. Theinventive compounds also can be used as surrogate markers to assessanti-amyloid therapies.

Imaging Techniques

One method of this invention determines the presence and location ofamyloid deposits in an organ or body area, such as the brain, of apatient. The method comprises administration of a detectable quantity ofa pharmaceutical composition containing an amyloid binding compound ofthe present invention called a “detectable compound,” or apharmaceutically acceptable water-soluble salt thereof, to a patient. A“detectable quantity” means that the amount of the detectable compoundthat is administered is sufficient to enable detection of binding of thecompound to amyloid. An “imaging effective quantity” means that theamount of the detectable compound that is administered is sufficient toenable imaging of binding of the compound to amyloid.

The invention employs amyloid imaging agents which, in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), or gamma imaging such as positronemission tomography (PET) or single-photon emission computed tomography(SPECT), are used to quantify amyloid deposition in vivo. The methodinvolves imaging a patient to establish a baseline of amyloiddeposition. For gamma imaging, the radiation emitted from the organ orarea being examined is measured and expressed either as total binding oras a ratio in which total binding in one tissue is normalized to (forexample, divided by) the total binding in another tissue of the samesubject during the same in vivo imaging procedure. Total binding in vivois defined as the entire signal detected in a tissue by an in vivoimaging technique without the need for correction by a second injectionof an identical quantity of labeled compound along with a large excessof unlabeled, but otherwise chemically identical compound. The methodcan further comprise at least one imaging session of a patient followingadministration of an anti-amyloid therapy. The method can also compriseimaging a patient before and after treatment with at least oneanti-amyloid agent. Imaging can be performed at any time during thetreatment.

For purposes of in vivo imaging, the type of detection instrumentavailable is a major factor in selecting a given label. For instance,radioactive isotopes and ¹⁹F can be used for in vivo imaging in themethods of the present invention. The type of instrument used will guidethe selection of the radionuclide or stable isotope. For instance, theradionuclide chosen must have a type of decay detectable by a given typeof instrument. Another consideration relates to the half-life of theradionuclide. The half-life should be long enough so that it is stilldetectable at the time of maximum uptake by the target, but short enoughso that the host does not sustain deleterious radiation. Theradiolabeled compounds of the invention can be detected using gammaimaging wherein emitted gamma irradiation of the appropriate wavelengthis detected. Methods of gamma imaging include, but are not limited to,SPECT and PET. In one embodiment, such as for SPECT detection, thechosen radiolabel will lack a particulate emission, but will produce alarge number of photons in a 140-200 keV range. For PET detection, theradiolabel will be a positron-emitting radionuclide such as ¹⁹F whichwill annihilate to form two 511 keV gamma rays that can be detected bythe PET camera.

In the present invention, amyloid binding compounds/imaging agents aremade which are useful for in vivo imaging and quantification of amyloiddeposition. These compounds are to be used in conjunction withnon-invasive neuroimaging techniques such as magnetic resonancespectroscopy (MRS) or imaging (MRI), positron emission tomography (PET),and single-photon emission computed tomography (SPECT). In accordancewith this invention, the benzofuran compounds may be labeled with ¹⁹F or¹³C for MRS/MRI by general organic chemistry techniques known to theart. For example, see ADVANCED ORGANIC CHEMISTRY: REACTION, MECHANISMS,AND STRUCTURE, 3^(rd) ed. (1985). The benzofuran compounds also may beradiolabeled with ¹⁸F, ¹¹C, ⁷⁵Br, or ⁷⁶Br for PET by techniques wellknown in the art and are described by Fowler, J. and Wolf, A. inPOSITRON EMISSION TOMOGRAPHY AND AUTORADIOGRAPHY 391-450 (Raven Press,1986). The benzofuran compounds also may be radiolabeled with ¹²³I forSPECT by any of several techniques known to the art. See, e.g.,Kulkarni, Int. J. Rad. Appl. & Inst. (Part B) 18: 647 (1991). Inaddition, the benzofuran compounds may be labeled with any suitableradioactive iodine isotope, such as, but not limited to ¹³¹I, ¹²⁵I, or¹²³I, by iodination of a diazotized amino derivative directly via adiazonium iodide, see Greenbaum, F. Am. J Pharm. 108: 17 (1936), or byconversion of the unstable diazotized amine to the stable triazene, orby conversion of a non-radioactive halogenated precursor to a stabletri-alkyl tin derivative which then can be converted to the iodocompound by several methods well known to the art. See, Satyamurthy andBarrio, J. Org. Chem. 48: 4394 (1983), Goodman et al., J. Org. Chem. 49:2322 (1984), and Mathis et al., J. Labell. Comp. and Radiopharm. 1994:905; Chumpradit et al., J. Med. Chem. 34: 877 (1991); Zhuang et al., J.Med. Chem. 37: 1406 (1994); Chumpradit et al., J. Med. Chem. 37: 4245(1994). For example, a stable triazene or tri-alkyl tin derivative ofbenzofuran is reacted with a halogenating agent containing ¹³¹I, ¹²⁵I,¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F or ¹⁹F. Thus, the stable tri-alkyl tin derivativesof benzofuran are novel precursors useful for the synthesis of many ofthe radiolabeled compounds within the present invention. As such, thesetri-alkyl tin derivatives are contemplated as one embodiment of thisinvention.

The benzofuran compounds also may be radiolabeled with known metalradiolabels, such as, for example, Technetium-99m (^(99m)Tc).Modification of the substituents to introduce ligands that bind suchmetal ions can be effected without undue experimentation by one ofordinary skill in the radiolabeling art. The metal radiolabeledbenzofuran can then be used to detect amyloid deposits. Preparingradiolabeled derivatives of Tc^(99m) is well known in the art. See, forexample, Zhuang et al., “Neutral and stereospecific Tc-99m complexes:[99mTc]N-benzyl-3,4-di-(N-2-mercaptoethyl)-amino-pyrrolidines (P-BAT)”Nuclear Medicine & Biology 26(2):217-24, (1999); Oya et al., “Small andneutral Tc(v)0 BAT, bisaminoethanethiol (N2S2) complexes for developingnew brain imaging agents” Nuclear Medicine & Biology 25(2):135-40,(1998); and Hom et al., “Technetium-99m-labeled receptor-specificsmall-molecule radiopharmaceuticals: recent developments and encouragingresults” Nuclear Medicine & Biology 24(6):485-98, (1997).

The methods of the present invention can use isotopes that aredetectable by nuclear magnetic resonance spectroscopy for purposes of invivo imaging and spectroscopy. Useful elements in magnetic resonancespectroscopy include but are not limited to ¹⁹F and ¹³C.

Suitable radioisotopes for purposes of this invention can bebeta-emitters, gamma-emitters, positron-emitters, and x-ray emitters.These radioisotopes include ¹³¹I, ¹²³I, ¹⁸F, ¹¹C, ⁷⁵Br, and ⁷⁶Br.Suitable stable isotopes for use in Magnetic Resonance Imaging (MRI) orSpectroscopy (MRS), according to this invention, include ¹⁹F and ¹³C.Suitable radioisotopes for in vitro quantification of amyloid inhomogenates of biopsy or post-mortem tissue include ¹²⁵I, ¹⁴C, and ³H.Exemplary radiolabels are ¹¹C or ¹⁸F for use in PET in vivo imaging,¹²³I for use in SPECT imaging, ¹⁹F for MRS/MRI, and ³H or ¹⁴C for invitro studies. However, any conventional method for visualizingdiagnostic imaging agents can be utilized in accordance with thisinvention.

According to one embodiment of the invention which relates to a methodof detecting amyloid deposits in biopsy or post-mortem tissue, themethod involves incubating formalin-fixed tissue with a solution of abenzofuran amyloid binding compound of the present invention. In oneembodiment, the solution is 25-100% ethanol, (with the remainder beingwater) saturated with a benzofuran amyloid binding compound according tothe present invention. Upon incubation, the compound stains or labelsthe amyloid deposit in the tissue, and the stained or labeled depositcan be detected or visualized by any standard method. Such detectionmeans include microscopic techniques such as bright-field, fluorescence,laser-confocal and cross-polarization microscopy.

The method of quantifying the amount of amyloid in biopsy or post-mortemtissue involves incubating a labeled derivative of benzofuran accordingto the present invention, or a water-soluble, non-toxic salt thereof,with homogenate of biopsy or post-mortem tissue. The tissue is obtainedand homogenized by methods well known in the art. In one embodiment, thelabel is a radiolabel, although other labels such as enzymes,chemiluminescent and immunofluorescent compounds are well known toskilled artisans. Exemplary radiolabels include but are not limited to¹²⁵I, ¹⁴C and ³H which are contained in a substituent substituted on oneof the compounds of the present formulae described herein. Tissuecontaining amyloid deposits will bind to the labeled derivatives of thebenzofuran amyloid binding compounds of the present invention. The boundtissue is then separated from the unbound tissue by any mechanism knownto the skilled artisan, such as filtering. The bound tissue can then bequantified through any means known to the skilled artisan. The units oftissue-bound radiolabeled benzofuran derivative then can be converted tounits of micrograms of amyloid per 100 mg of tissue by comparison to astandard curve generated by incubating known amounts of amyloid with theradiolabeled benzofuran derivative.

The method of distinguishing an Alzheimer's diseased brain from a normalbrain comprises obtaining tissue from (a) the cerebellum and (b) anotherarea of the same brain, other than the cerebellum, from normal subjectsand from subjects suspected of having Alzheimer's disease. Such tissuesare made into separate homogenates using methods well known to theskilled artisan, and then are incubated with a radiolabeled benzofuranamyloid binding compound of formula (I). The amount of tissue whichbinds to the radiolabeled benzofuran amyloid binding compound is thencalculated for each tissue type (e.g. cerebellum, non-cerebellum,normal, abnormal) and the ratio for the binding of non-cerebellum tocerebellum tissue is calculated for tissue from normal and for tissuefrom patients suspected of having Alzheimer's disease. These ratios arethen compared. If the ratio from the brain suspected of havingAlzheimer's disease is above 90% of the ratios obtained from normalbrains, the diagnosis of Alzheimer's disease is made. The normal ratioscan be obtained from previously obtained data, or alternatively, can berecalculated at the same time the suspected brain tissue is studied.

The ability of the present compounds to specifically bind toneurofibrially tangles over amyloid plaques is particularly evident atconcentrations less than 10 nM, which includes the in vivo concentrationrange of PET radiotracers. At these low concentrations, which containsonly tangles and no plaques, significant binding does not result whencompared to control brain tissue containing neither plaques nor tangles.However, incubation of homogenates of brain tissue that contains mainlyplaques and some tangles with radiolabeled compounds of the formulaedescribed herein, results in a significant increase in binding whencompared to control tissue without plaques or tangles. This datasuggests that one advantage of these compounds is their specificity forAβ deposits at concentrations less than 10 nM. These low concentrationsare detectable in PET studies, making PET detection using radiolabeledcompounds of the formulae herein described which are specific for Aβdeposits possible. The use of such compounds permits PET detection in Aβdeposits such as those found in plaques and cerebrovascular amyloid.Since it has been reported that Aβ levels in the frontal cortex areincreased prior to tangle formation, this would suggest thatradiolabeled compounds of the present invention, used as PET tracers,would be specific for the earliest changes in AD cortex. Naslund et al.,JAMA 283: 1571 (2000).

Method and Use for Detecting Amyloid Deposit(s) In Vivo

As mentioned above, the invention further provides, in one embodiment, amethod for detecting amyloid deposit(s) in vivo, comprising:

(i) administering to an animal an effective amount of compound accordingto formula (I), wherein the compound would bind to any amyloiddeposit(s) in the animal; and

(ii) detecting binding of the compound to amyloid deposit(s) in theanimal.

After a sufficient time has elapsed for the compound to bind with theamyloid deposit(s), for example 30 minutes to 48 hours followingadministration, the binding may be detected by any means known in theart. Examples of detection means include, without limitation, assays(such as immunometric, calorimetric, densitometric, spectrographic andchromatographic assays), non-invasive neuroimaging techniques (such asmagnetic resonance spectroscopy (MRS), magnetic resonance imaging (MRI),and gamma imaging techniques such as single-photon emission computedtomography (SPECT) and positron emission tomography (PET). For gammaimaging, the radiation emitted from the organ or area being examined ismeasured and expressed either as total binding or as a ratio in whichtotal binding in one tissue is normalized to (for example, divided by)the total binding in another tissue of the same subject during the samein vivo imaging procedure. Total binding in vivo is defined as theentire signal detected in a tissue by an in vivo imaging techniquewithout the need for correction by a second injection of an identicalquantity of labeled compound along with a large excess of unlabeled, butotherwise chemically identical compound.

The type of detection instrument available can be a factor in selectingthe radioactive halo or carbon isotope. For instance, the selectedradioisotope should have a type of decay that is detectable by a giveninstrument. Another consideration relates to the half-life of theradioisotope. The half-life should be long enough such that theradioisotope is still detectable at the time of maximum uptake by thetarget, but short enough such that the host does not sustain deleteriousradiation. For SPECT detection, the selected radioisotope may lack aparticulate emission, but may produce a large number of photons in the140-200 keV range. For PET detection, the selected radioisotope may be apositron-emitting radioisotope, which annihilates to form two 511 keVgamma rays detectable by a PET camera.

Useful radioisotopes include, without limitation: ¹²⁵I, ¹⁴C, and ³H forin vitro quantification of amyloid in homogenates of biopsy orpost-mortem tissue; ¹¹C and ¹⁸F for PET in vivo imaging; ¹²³I for SPECTimaging; ¹⁸F for MRS/MRI; ³H or ¹⁴C for in vitro studies; and ¹⁸F and¹³C for magnetic resonance spectroscopy. In one embodiment, thedetecting is effected by gamma imaging, magnetic resonance imaging ormagnetic resonance spectroscopy. In another embodiment, the gammaimaging is PET or SPECT.

Method and Use for Detecting Amyloid Deposit(s) In Vitro

This invention further provides a method for detecting amyloiddeposit(s) in vitro comprising:

(i) contacting a bodily tissue with an effective amount of a compoundaccording to formula (I), wherein the compound would bind any amyloiddeposit(s) in the tissue; and

(ii) detecting binding of the compound to amyloid deposit(s) in thetissue.

The binding may be detected by any means known in the art. Examples ofdetection means include, without limitation, microscopic techniques,such as bright-field, fluorescence, laser-confocal andcross-polarization microscopy.

In one embodiment, the tissue is biopsy or post-mortem tissue that isformalin-fixed or fresh-frozen. In another embodiment, the tissue ishomogenized. In yet another embodiment, the inventive compound is in asolution that further comprises 25-99% ethanol, with the remainder ofthe solution being water. In yet another embodiment, the solutioncomprises 0-50% ethanol and 0.0001 to 100 μM of the compound. In yetanother embodiment, the method further comprises (iii) separating fromthe tissue the amyloid deposit(s) bound to the compound; and (iv)quantifying the amyloid deposit(s) bound to the inventive compound. Thebound amyloid deposit(s) may be separated from the tissue by any meansknown in the art, such as filtering. The amount of bound amyloiddeposit(s) may be converted to units of μg of amyloid deposit(s) per 100mg of tissue by comparison to a standard curve generated by incubatingknown amounts of amyloid with the inventive compound or pharmaceuticallyacceptable salt, hydrate, solvate or prodrug.

Method and Use for Distinguishing Alzheimer's Diseased Brain from NormalBrain

This invention further provides a method for distinguishing anAlzheimer's diseased brain from a normal brain comprising:

(i) obtaining tissues from (a) the cerebellum and (b) another area ofthe same brain, of a normal animal and of an animal suspected of havingAlzheimer's disease;

(ii) contacting the tissues with a compound according to formula (I);

(iii) quantifying the amyloid bound to the compound;

(iv) calculating the ratio of the amount of amyloid in the area of thebrain other than the cerebellum to the amount of amyloid in thecerebellum;

(v) comparing the ratio for a normal animal with the ratio for an animalsuspected of having Alzheimer's disease.

A diagnosis of Alzheimer's disease may be made if the ratio for ananimal suspected of having Alzheimer's disease is, for example, above90% of the ratio for a normal animal. For this method, a “normal” animalis one that is not suffering from Alzheimer's disease.

Administration and Pharmaceutical Compositions

According to the present invention, a pharmaceutical compositioncomprising an amyloid imaging agent of formula (I) can be administeredto subjects in whom amyloid or amyloid fibril formation are anticipated,e.g., patients clinically diagnosed with Alzheimer's disease or anotherdisease associated with amyloid deposition.

Administration to the subject can be local or systemic and accomplished,for example, intravenously, intraarterially, intrathecally (via thespinal fluid) or the like. Administration also can be intradermal orintracavitary, depending upon the body site under examination. After asufficient time has elapsed for the compound to bind with the amyloid,for example 30 minutes to 48 hours, the area of the subject underinvestigation is examined by routine imaging techniques such as MRS/MRI,SPECT, planar scintillation imaging, PET, and any emerging imagingtechniques, as well. The exact protocol will necessarily vary dependingupon factors specific to the patient, as noted above, and depending uponthe body site under examination, method of administration and type oflabel used; the determination of specific procedures would be routine tothe skilled artisan. For brain imaging, as one example, the amount(total or specific binding) of the bound radioactively labeledbenzofuran compound or analogue of the present invention is measured andcompared (as a ratio) with the amount of labeled benzofuran compoundbound to the cerebellum of the patient. This ratio is then compared tothe same ratio in age-matched normal brain. For organ imaging, asanother example, the amount (total or specific binding) of the boundradioactively labeled thioflavin derivative or analogue of the presentinvention is measured and compared (as a ratio) with the amount oflabeled thioflavin derivative bound to the organ of the patient. Thisratio is then compared to the same ratio in age-matched normal organ.

The amyloid imaging agents of the present invention can be administeredin the form of injectable compositions, as noted above, but may also beformulated into well known drug delivery systems (e.g., oral, rectal,parenteral (intravenous, intramuscular, or subcutaneous),intracisternal, intravaginal, intraperitoneal, local (powders, ointmentsor drops), or as a buccal or nasal spray). A typical composition forsuch purpose comprises a pharmaceutically acceptable carrier. Forinstance, the composition may contain about 10 mg of human serum albuminand from about 0.5 to 500 micrograms of the labeled benzofuran compoundper milliliter of phosphate buffer containing NaCl. Otherpharmaceutically acceptable carriers include aqueous solutions,non-toxic excipients, including salts, preservatives, buffers and thelike, as described, for instance, in REMINGTON′S PHARMACEUTICALSCIENCES, 15th Ed. Easton: Mack Publishing Co. pp. 1405-1412 and1461-1487 (1975) and THE NATIONAL FORMULARY XIV., 14th Ed. Washington:American Pharmaceutical Association (1975).

Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oil and injectable organic esters such as ethyloleate. Aqueous carriers include water, alcoholic/aqueous solutions,saline solutions, parenteral vehicles such as sodium chloride, Ringer'sdextrose, etc. Intravenous vehicles include fluid and nutrientreplenishers. Preservatives include antimicrobial, anti-oxidants,chelating agents and inert gases. The pH and exact concentration of thevarious components the pharmaceutical composition are adjusted accordingto routine skills in the art. See, Goodman and Gilman's THEPHARMACOLOGICAL BASIS FOR THERAPEUTICS (7th Ed.).

The PET scanning protocol can comprise a standard whole body scan(covering from head to pelvis) completed 15-60 min after the injectionof the radiopharmaceutical or a scan over a particular body area (e.g.,heart, lungs, liver, kidneys). This scanning protocol is analogous to awhole body or a focused body area PET oncology scan performed with[F-18]2-fluoro-2-deoxyglucose (FDG). That is, the amyloid-specificradiopharmaceutical is injected intravenously, time is alloted forradiotracer distribution throughout the body, radiotracer uptake in theorgan(s) of interest, and clearance from the blood and other organs inwhich amyloid is absent, and a 20-40 min scan is performed over thewhole body or over a particular body area to image amyloid-boundradiotracer. In addition, the imaging scan(s) can be used tosubsequently direct biopsy sampling of the scanned tissue(s).

Generally, the dosage of the detectably labeled benzofuran compoundaccording to formula (I) will vary depending on considerations such asage, condition, sex, and extent of disease in the patient,contraindications, if any, concomitant therapies and other variables, tobe adjusted by a physician skilled in the art. Dose levels on the orderof about 0.001 μg/kg/day to about 10,000 mg/kg/day of an inventivecompound are useful for the inventive methods. In one embodiment, thedose level is about 0.001 μg/kg/day to about 10 μg/kg/day. In anotherembodiment, the dose level is about 0.01 μg/kg/day to about 1.0μg/kg/day. In yet another embodiment, the dose level is about 0.1mg/kg/day to about 100 mg/kg/day.

The specific dose level for any particular patient will vary dependingupon various factors, including the activity and the possible toxicityof the specific compound employed; the age, body weight, general health,sex and diet of the patient; the time of administration; the rate ofexcretion; the drug combination; and the form of administration.Typically, in vitro dosage-effect results provide useful guidance on theproper doses for patient administration. Studies in animal models arealso helpful. The considerations for determining the proper dose levelsare well known in the art and within the skills of an ordinaryphysician.

Any known administration regimen for regulating the timing and sequenceof drug delivery may be used and repeated as necessary to effecttreatment in the inventive methods. The regimen may include pretreatmentand/or co-administration with additional therapeutic agent(s).

In one embodiment, the compounds according to formula (I) areadministered to an animal that is suspected of having or that is at riskof developing a disease, disorder or condition characterized by amyloiddeposition. For example, the animal may be an elderly human.

In another embodiment, the inventive compounds bind to Aβ with adissociation constant (K_(D)) of about 0.0001 μM to about 10.0 μM whenmeasured by binding to synthetic Aβ peptide or AD brain tissue.

This invention further provides a pharmaceutical composition comprising:

(i) an effective amount of at least one inventive compound; and

(ii) a pharmaceutically acceptable carrier.

The composition may comprise one or more additional pharmaceuticallyacceptable ingredient(s), including without limitation one or morewetting agent(s), buffering agent(s), suspending agent(s), lubricatingagent(s), emulsifier(s), disintegrant(s), absorbent(s), preservative(s),surfactant(s), colorant(s), flavorant(s), sweetener(s) and therapeuticagent(s).

The composition can be formulated into solid, liquid, gel or suspensionform for: (1) oral administration as, for example, a drench (aqueous ornon-aqueous solution or suspension), tablet (for example, targeted forbuccal, sublingual or systemic absorption), bolus, powder, granule,paste for application to the tongue, hard gelatin capsule, soft gelatincapsule, mouth spray, emulsion and microemulsion; (2) parenteraladministration by subcutaneous, intramuscular, intravenous or epiduralinjection as, for example, a sterile solution, suspension orsustained-release formulation; (3) topical application as, for example,a cream, ointment, controlled-release patch or spray applied to theskin; (4) intravaginal or intrarectal administration as, for example, apessary, cream or foam; (5) sublingual administration; (6) ocularadministration; (7) transdermal administration; or (8) nasaladministration.

In one embodiment, the composition can be formulated for intravenousadministration and the carrier includes a fluid and/or a nutrientreplenisher. In another embodiment, the composition is capable ofbinding specifically to amyloid in vivo, is capable of crossing theblood-brain barrier, is non-toxic at appropriate dose levels and/or hasa satisfactory duration of effect. In yet another embodiment, thecomposition comprises about 10 mg of human serum albumin and from about0.5 to 500 mg of the inventive compound per milliliter of phosphatebuffer containing NaCl.

In addition, the present benzofuran compounds can be used in a methodfor determining the efficacy of therapy in the treatment of amyloidosis.The method involves the use of amyloid imaging as a surrogate marker.Surrogate markers are a special type of biomarker that may be used inplace of clinical measurements as a clinical endpoint for drug approvalpurposes. For example, the measurement of cholesterol levels is now anaccepted surrogate marker of atherosclerosis. The present inventioninvolves the use of amyloid imaging as a surrogate marker of efficacyfor anti-amyloid therapies.

The present method provides a means of evaluating success ofanti-amyloid therapies. In some embodiments, the present method providesa means for evaluating clinical success of anti-amyloid therapies. Insome embodiments, the method may be used to evaluate clinical success inmildly impaired subjects with few or no clinical symptoms to follow. Thebasic method of determining the efficacy of therapy in the treatment ofamyloidosis involves:

(A) administering to a patient in need thereof an effective amount ofcompound of formula (I) or a pharmaceutically acceptable salt asdecribed above:

(B) imaging said patient; then

(C) administering to said patient in need thereof at least oneanti-amyloid agent;

(D) subsequently administering to said patient in need thereof aneffective amount of a compound of formula (I);

(E) imaging said patient; and

(F) comparing levels of amyloid deposition in said patient beforetreatment with at least one anti-amyloid agent to levels of amyloiddeposition in said patient after treatment with at least oneanti-amyloid agent.

The detectable label includes any atom or moiety which can be detectedusing an imaging technique known to those skilled in the art. Typically,the detectable label is selected from the group consisting of ³H, ¹³¹I,¹²⁵I, ¹²³I, ⁷⁶Br, ⁷⁵Br, ¹⁸F, CH₂—CH₂—X*, O—CH₂—CH₂—X*, CH₂—CH₂—CH₂—X*,O—CH₂—CH₂—CH₂—X* (wherein X*=¹³¹I, ¹²³I, ⁷⁶Br, ⁷⁵Br or ¹⁸F), ¹⁹F, ¹²⁵I,a carbon-containing substituent selected from the group consisting oflower alkyl, (CH2)nOR′, CF₃, CH₂—CH₂X, O—CH₂—CH₂X, CH₂—CH₂—CH₂X,O—CH₂—CH₂—CH₂X (wherein X═F, Cl, Br or I), CN, (C═O)—R′, (C═O)N(R′)₂,O(CO)R′, COOR′, CR′═CR′—R_(ph) and CR₂′—CR₂′—R_(ph) wherein at least onecarbon is ¹¹C, ¹³C or ¹⁴C and a chelating group (with chelated metalgroup) of the form W-L* or V-W-L*, wherein V is selected from the groupconsisting of —COO—, —CO—, —CH₂O— and —CH₂NH—; W is —(CH₂)_(n) wheren=0,1,2,3,4, or 5; and L* is:

wherein M* is ^(99m)Tc. In one embodiment, the detectable label is aradiolabel.

Anti-Amyloid Therapies and Uses

Another embodiment of the invention is a method for determining theefficacy of therapy in the treatment of amyloidosis a patient in needthereof. The method comprises administering a compound of formula (I)and then imaging the patient. After the imaging, at least oneanti-amyloid agent/anti-amyloid therapy is administered to the patient.The amount administered, the route of administration, and the durationof therapy are determined by one skilled in the art based on age,weight, and condition of the patient. Such determinations are within thepurview of the skilled practitioner. Suitable amounts include, but arenot limited to, 0.01 to 100 mg/kg. Suitable routes of administrationinclude, but are not limited to oral, subcutaneous and intravenous.Suitable durations of therapy include, but are not limited to one singledose to four doses per day given indefinitely. Suitable times to imageinclude, but are not limited to immediately after the first dose to tenyears after the most recent dose. Exemplary times to image include butare not limited to those between 7 days and 6 months after the mostrecent dose.

An “anti-amyloid agent” or an “anti-amyloid therapy” is any agent orcombination of agents that treat or prevent amyloidosis. Examples ofdiseases associated with amyloid deposition, amyloidosis, includeAlzheimer's Disease, Down's Syndrome, Type 2 diabetes mellitus,hereditary cerebral hemorrhage amyloidosis (Dutch), amyloid A(reactive), secondary amyloidosis, MCI, familial mediterranean fever,familial amyloid nephropathy with urticaria and deafness (Muckle-wellsSyndrome), amyloid lambda L-chain or amyloid kappa L-chain (idiopathic,myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish)), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, systemic senile amyloidoses, AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-I (familial amyloidoticpolyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice),fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, CreutzfeldJacob disease, Gertsmann-Straussler-Scheinker syndrome, bovinespongiform encephalitis) or in cases of persons who are homozygous forthe apolipoprotein E4 allele, and the condition associated withhomozygosity for the apolipoprotein E4 allele or Huntington's disease.The invention contemplates diseases associated with amyloid plaquedeposition. In one embodiment, the disease associated with amyloiddeposition is AD.

The present benzofurans according to formula (I) can be used for amyloidimaging serving as a surrogate marker of efficacy for anti-amyloidtherapy. Administration of an amyloid imaging agent to establish abaseline of amyloid deposition and subsequent imaging of a patient bothbefore and after treatment of the patient with an anti-amyloid agentallows for determination of the efficacy of the anti-amyloid therapy.The method can be used to determine the efficacy of any anti-amyloidtreatment because an amyloid imaging agent can be administered, and thepatient can be imaged, before and after any anti-amyloid therapy. Themethod contemplates determining anti-amyloid therapies which areineffective for treating diseases associated with amyloid deposition, aswell as anti-amyloid therapies which are effective for treating diseasesassociated with amyloid deposition. A person of ordinary skill in theart can determine the conditions and dosing of the anti-amyloid therapyaccording to appropriate protocols. Therefore, the present inventioncontemplates determining the efficacy of anti-amyloid therapies that arenow known, as well as therapies that are yet to be discovered. Exemplarynon-limiting anti-amyloid therapies are described below.

In some embodiments, the efficacy of acetylcholinesterase inhibitors inthe treatment of amyloidosis can be determined by the present method.Acetylcholinesterase therapy is based on studies of degenerationpatterns in AD which identified substantial decreases among groups ofneurons in the basal forebrain. These cells all used the transmitteracetylcholine, and their loss meant that less acetylcholine was beingreleased at their former terminals in the cortex. Several drugs, such astacrine, donepezil, rivastigmine and galantamine have been developedbased on these findings, and are hypothesized to work by inhibiting theenzyme acetylcholinesterase (Ingram, V., American Scientist, 2003,91(4):312-321).

In other embodiments, the efficacy of anti-amyloid therapy targetingenzymes responsible for formation of noxious fragments of amyloidprecursor protein (APP) in the treatment of amyloidosis is determined bythe inventive compounds according to the described methodology. In someembodiments, the noxious fragments of the amyloid precursor protein(APP) is misfolded Aβ peptide. For example, the overproduction of Aβ1-42fragment is considered by some scientists to be a root cause of AD. TheAβ1-42 fragment is formed by cleavage of APP by the (3-secretase enzyme(BACE1) (which produces the amino terminus) and the γ-secretase enzyme(which cleaves the carboxyl terminus of APP). Inhibitors of thesesecretase enzymes may be used as anti-amyloid therapies (Ingram, V.,American Scientist, 2003, 91(4):312-321).

In some embodiments, the efficacy of immunotherapeutic strategies in thetreatment of amyloidosis can be determined by the present method.Immunotherapy works by using the patient's immune system to locate anddestroy amyloid plaques and many immunotherapy strategies are beingactively pursued by scientists. The immunotherapeutic strategies can beeither passive or active. For example, in active immunotherapy, apatient may receive an injection or nasal-spray application of the Aβpeptide, leading to an anti-amyloid immune response. Passiveimmunotherapy, on the other hand, might involve bypassing the betaamyloid protein, using instead antiserum that has already been producedin response to beta amyloid. Immunotherapy, involving antibodies againstAβ peptide, has been studied for the treatment of AD. For example,AN-1792 is a preparation of preaggregated synthetic amyloid-beta (Aβ;1-42 length) along with QS-21 adjuvant (Hock, C. et al., 2003, Neuron,38:547-554). Approximately 300 AD patients have been treated with thispreparation prior to suspension of the clinical trial due to sideeffects (Birmingham, K. and Frantz, S., 2002, Nature Medicine,8:199-200).

In other embodiments, the efficacy of neuroprotective strategies in thetreatment of amyloidosis is determined by the present method. Forexample, many clinicians recommend that patients take high doses(1000-2000 IU/day) of vitamin E. Other types of neuroprotectivestrategies that have been suggested for the treatment of amyloidosis arehigh doses of vitamin C, calcium channel modulators, free-radicalscavengers, and metal ion chelators (Selkoe, et al., Annu. Rev.Pharmacol. Toxicol., 2003, 43:545-84).

In some embodiments, the efficacy of anti-inflammatory drugs (NSAIDs)strategies in the treatment of amyloidosis is determined by the presentmethod. Treatments involving NSAIDs are based on evidence that acellular inflammatory response in the cortex is elicited by theprogressive accumulation of Aβ peptide. Exemplary anti-inflammatorydrugs are prednisone, nonspecific cyclooxygenase inhibitors, andcyclooxygenase-2 inhibitors. (Clark, M., et al., Annals of InternalMedicine, 2003, 138(5):400-410; and Hardy, John, Annu. Rev. Med., 2004,55:15-25).

In some embodiments, the present method can determine the efficacy ofcholesterol-lowering therapies including, but are not limited to, the3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors (statins).Treatments involving cholesterol-lowering drugs (such as statins) arebased on epidemiological evidence that patients treated with statinshave a lower incidence of AD and that statins can alter the metabolismof Aβ to decrease AR levels (Wolozin, B (2002) Cholesterol andAlzheimer's disease. Biochemical Society Transactions. 30:525-529).Exemplary cholesterol-lowering statin drugs include lovastatin,pravastatin, rosuvastatin, fluvastatin, atorvastatin and simvastatin.Other cholesterol-lowering drugs include niacin, cholestyramine,fenofibrate, colesevelam and ezetimibe.

In other embodiments, the efficacy of small molecules that eliminate theneurotoxicity of the aggregated Aβ-42 in the treatment of amyloidosis isdetermined by the present method. Such a drug, when administered, forexample, early in disease progression, would “detoxify” the graduallyaccumulating Aβ peptide before any permanent damage is inflicted on theneurons. (Clark, M., et al., Annals of Internal Medicine, 2003,138(5):400-410)

In some embodiments, the efficacy of “decoy peptides” in the treatmentof amyloidosis is determined by the present method. Decoy peptides aresmall molecules that bind to the aggregating Aβ-42 peptide and force itto assume a nontoxic structure. Exemplary decoy peptides are smallpeptides (5, 6 or 9 amino acids long), selected from large libraries ofprotein fragments by their ability to form a tight association withtagged Aβ-42. Clark, M., et al., Annals of Internal Medicine, 2003,138(5):400-410).

In other embodiments, the efficacy of cholesterol homeostasis modulationin the treatment of amyloidosis is determined by the present method.Chronic use of cholesterol-lowering drugs has recently been associatedwith a lower incidence of AD. Concurrently, high-cholesterol diets havebeen shown to increase Aβ pathology in animals, and cholesterol-loweringdrugs have been shown to reduce pathology in APP transgenic mice.Clinical trials are underway to study the effect of cholesterolhomeostasis modulation in the treatment of AD. (Hardy, John, Annu. Rev.Med., 2004, 55:15-25)

Certain antibodies such as the one termed m266 (DeMattos, R B, Bales, KR, Cummins, D J, Dodart, J C, Paul, S M, Holtzman, D M (2001)“Peripheral anti-A beta antibody alters CNS and plasma A beta clearanceand decreases brain A beta burden in a mouse model of Alzheimer'sdisease” Proc. Natl. Acad. Sci. USA 98:8850-8855) or molecules otherthan antibodies (Matsuoka, Y, Saito, M, LaFrancois, J, Saito, M, Gaynor,K, Olm, V, Wang, L, Casey, E, Lu, Y, Shiratori, C, Lemere, C, Duff, K(2001) “Novel therapeutic approach for the treatment of Alzheimer'sdisease by peripheral administration of agents with an affinity tobeta-amyloid” Journal of Neuroscience, 23:29-33) are believed to lowerbrain amyloid by binding to Aβ peptides in the blood, thereby creating a“peripheral sink” and shifting the equilibrium of Aβ from the brain tothe blood, where it can be cleared from the body. Such agents arereferred to herein as “peripheral sink agents.”

Evaluating The Efficacy Of The Anti-Amyloid Therapy

The methodology employing the benzofurans according to formula (I) fordetermining the efficacy of therapy in the treatment of amyloidosiscomprises administering to a patient in need thereof a compound offormula (I) and imaging the patient. After imaging, at least oneanti-amyloid agent is administered to the patient. Then, an effectiveamount of a compound of formula (I) is administered to the patient andthe patient is imaged again. Finally, baseline levels of amyloiddeposition in the patient before treatment with the anti-amyloid agentare compared with levels of amyloid deposition in the patient followingtreatment with the anti-amyloid agent. Such a comparison is within thepurview of a skilled practitioner.

In some embodiments, the levels of amyloid deposition in the patientbefore treatment with the anti-amyloid agent will be higher than thelevels of amyloid deposition in the patient following treatment with theanti-amyloid agent. Such a result indicates that the anti-amyloidagent/anti-amyloid therapy is effective in the treatment of diseasesassociated with amyloid deposition.

For example, AN-1792 is a preparation of preaggregated syntheticamyloid-beta (Aβ; 1-42 length) along with QS-21 adjuvant. Approximately300 AD patients have been treated with this preparation prior tosuspension of the clinical trial due to side effects (Birmingham, K. andFrantz, S., 2002, Nature Medicine, 8:199-200). Despite this set back,optimism over this approach has been raised by two findings. First, inthe only autopsy report yet published regarding an AN-1792-treated ADpatient, there were several unusual findings including: (i) extensiveareas of neocortex with very few Aβ plaques; (ii) areas of cortex thatwere devoid of Aβ plaques contained densities of tangles, neuropilthreads and cerebral amyloid angiopathy (CAA) similar to unimmunized AD,but lacked plaque-associated dystrophic neurites and astrocyte clusters;(iii) in some regions devoid of plaques, Aβ-immunoreactivity wasassociated with microglia (Nicoll, J. et al., 2003, Nature Medicine9:448-452). Second, in a small subset of 30 AN-1792-treated patients,those patients who generated antibodies against Aβ, as determined by atissue amyloid plaque immunoreactivity (TAPIR) assay showedsignificantly slower rates of decline of cognitive functions andactivities of daily living, as indicated by the Mini Mental StateExamination, the Disability Assessment for Dementia, and the VisualPaired Associates Test of delayed recall from the Wechsler Memory Scale,as compared to patients without such antibodies (Hock, C. et al., 2003,Neuron, 38:547-554).

In another embodiment, the invention contemplates administering compoundsaccording to formula (I) to patienta in a method of imaging amyloiddeposits in the brains of patients who do not meet clinical criteria forthe diagnosis of AD. These include are but are not limited to patientspresenting with clinical signs of dementia or patients with a mildcognitive impairment, such as, for example, patients presenting adementing disorder of questionable etiology, where data from amyloidimaging of patients reveals that certain amyloid deposits are apremonitory symptom of AD or another amyloid deposition disorder.

Another embodiment of the present invention is a method of identifying apatient as prodromal to a standard clinical diagnosis of an amyloiddeposition disease. The method comprises the use of amyloid imagingagents to obtain quantitative and qualitative data from a patient.Quantitative and qualitative amyloid imaging, in accordance with thepresent invention, should allow for earlier and more accurate diagnosisof amyloid deposit diseases, and should aid in the development ofanti-amyloid therapies. The target patient for this methodology can be apatient presenting signs of clinical dementia or a patient exhibitingclinical signs of mild cognitive impairment.

In keeping with conventional practice, the practitioner may applydifferent criteria for a determination of signs of clinical dementia.Such criteria include but are not limited to Diagnostic and StatisticalManual of Mental Disorders, third edition (DSM-III) Alzheimer's DiseaseDiagnostic and Treatment Center (ADDTC), International StatisticalClassification of Diseases, 10^(th) Revision (ICD-10), NationalInstitute of Neurological Disorders and Stroke-AssociationInternationale pour la Recherche et l'Enseignment en Neurosciences(NINDS-AIREN) and Diagnostic and Statistical Manual of Mental Disorders,Fourth Edition (DSM-IV). See Pohjasvaara et al., Stroke 31: 2952-57(2000).

Clinical characterization of a patient as mild cognitive impairment iswell within the skill of the practitioner. The testing of a patient toelucidate such a condition involves performing a series of mental tests.The methods for clinical diagnosis are widely reviewed and arediscussed, e.g., in Petersen et al., Arch. Neurol. 56: 303-08 (1999).

Based on clinical testing alone, subjects identified with MCI mayconvert to a diagnosis of AD (at a rate of about 10-15% per year),remain MCI, or revert to a diagnosis of “normal” (10-15% per year).Larrieu et al., Neurology 59: 1594-99 (1926). Accordingly, there isconsiderable prognostic uncertainty associated with this clinicaldiagnosis. The ability to identify the presence or absence of brainamyloid deposition in a subject clinically diagnosed with MCI has thepotential to greatly increase the accuracy of prognosis for conversionto AD.

The category of diseases associated with amyloid deposition includes butis not limited to Alzheimer's Disease, Down's Syndrome, Type 2 diabetesmellitus, hereditary cerebral hemorrhage amyloidosis (Dutch), amyloid A(reactive), secondary amyloidosis, familial Mediterranean fever,familial amyloid nephropathy with urticaria and deafness (Muckle-wellsSyndrome), amyloid lambda L-chain or amyloid kappa L-chain (idiopathic,myeloma or macroglobulinemia-associated) A beta 2M (chronichemodialysis), ATTR (familial amyloid polyneuropathy (Portuguese,Japanese, Swedish)), familial amyloid cardiomyopathy (Danish), isolatedcardiac amyloid, systemic senile amyloidoses, AIAPP or amylininsulinoma, atrial naturetic factor (isolated atrial amyloid),procalcitonin (medullary carcinoma of the thyroid), gelsolin (familialamyloidosis (Finnish)), cystatin C (hereditary cerebral hemorrhage withamyloidosis (Icelandic)), AApo-A-I (familial amyloidoticpolyneuropathy-Iowa), AApo-A-II (accelerated senescence in mice),fibrinogen-associated amyloid; and Asor or Pr P-27 (scrapie, CreutzfeldJacob disease, Gertsmann-Straussler-Scheinker syndrome, bovinespongiform encephalitis) or in cases of persons who are homozygous forthe apolipoprotein E4 allele, and the condition associated withhomozygosity for the apolipoprotein E4 allele or Huntington's disease.In one embodiment, the disease associated with amyloid deposition is anamyloid plaque deposition disease. A specific disease associated withamyloid deposition is AD.

According to the invention, a basic methodology of identifying a patientas prodromal to an amyloid deposition disease entails:

(A) administering to the patient, who is presenting with signs ofclinical dementia or presenting with clinical signs of a mild cognitiveimpairment, in need thereof an effective amount of compound of formula Idescribed above or a pharmaceutically acceptable salt thereof;

(B) imaging said patient to obtain data and

(C) analyzing said data to ascertain amyloid levels in said patient withreference to a normative patient.

One embodiment relates to a method for diagnosing a patient presentingwith a dementing of questionable etiology. This method comprisesdetermining if dementias of questionable etiology are likely to be AD oranother amyloid deposition disorder based on the finding of amyloiddeposition. The method comprises administering to a patient a compoundof Formula (I), imaging the patient to obtain data and determining ifthe dementia of questionable etiology is AD based on the finding ofamyloid deposition.

The term “dementing disorder of questionable etiology” refers to thecondition in which a person presents for clinical evaluation (which mayconsist of neurological, psychiatric, medical and neuropsychologicalevaluations commonly employed by those skilled in the art of diagnosingpersons with dementing disorders) and, after that clinical evaluation,the evaluator finds evidence that some dementing disorder may be present(based on evidence of subjective memory complaints, description ofmemory complaints by informants familiar with the persons deviation fromnormal functioning, or poor performance on neuropsychological andclinical tests commonly used by those skilled in the art), but, can notfind sufficient evidence for any single clinically defined dementingdisorder (such as AD, frontotemporal dementia, Dementia with LewyBodies, Vascular dementia, pseudodementia due to Major Depression,Creutzfeld Jacob disease and others known to those skilled in the art)or finds that the person shows evidence of more than one singledementing disorder to the degree that the distinction between these two(or more) dementing disorders is questionable in this person.

This embodiment of the invention employs amyloid imaging agents which,in conjunction with non-invasive neuroimaging techniques such asmagnetic resonance spectroscopy (MRS) or imaging (MRI), or gamma imagingsuch as positron emission tomography (PET) or single-photon emissioncomputed tomography (SPECT), are used to quantify amyloid deposition invivo. These imaging techniques acquire data on many brain regions.Quantitation on specific regions is achieved by delineating “regions ofinterest or ROI”.

In accordance with this embodiment, data obtained from patients usingone of the imaging techniques mentioned above can be compared to datafrom normative patients with a conclusion based on criteria whichdistinguish the patient as prodromal to a standard clinical diagnosis ofan amyloid deposition disease.

Using the same protocol, one can compare data obtained from the imagingtechniques applied to the patients in order to:

define a dementing disorder of questionable etiology as being caused byan amyloid deposition disease;

distinguish Alzheimer's disease from frontotemporal dementia; monitor apatient to determine onset of Alzheimer's disease;

diagnose Alzheimer's disease in a patient clinically diagnosed with mildcognitive impairment;

identify a patient as prodromal to Alzheimer's disease;

identify a patient as having a disease associated with an amyloiddeposition disorder wherein the patient is presenting with a dementingdisorder of questionable etiology or identify a patient as havingAlzheimer's disease wherein the patient is presenting with a dementingdisorder of questionable etiology.

Data Analysis Of Amyloid Imaging

The data obtained can be quantitatively expressed in terms ofStandardized Uptake Value (SUV) or in terms of pharmacokinetic modelingparameters such as the Logan distribution volume ratio (DVR) to areference tissue such as cerebellum. Subjects who are more than onestandard deviation above the typical control value of SUV or DVR wouldbe considered to have a “positive” test and be considered to beprodromal to a clinical diagnosis of an amyloid deposition disease suchas AD. Specifically, subjects can be considered “positive” if their40-60 min average SUV is greater than 1.0 in frontal, parietal orposterior cingulate cortex. This value clearly separated AD patientsfrom controls in the initial human study (Klunk, et al., 2004, Ann.Neurol., 55(3):306-19). Likewise, subjects can be considered “positive”if their Logan DVR value exceeds 1.5 in frontal, parietal or posteriorcingulate cortex. These brain areas and exact cutoffs are given only asexamples and further work may disclose additional brain areas that areuseful and the cutoff values may be refined and other modelingtechniques (such as compartmental modeling, graphical analysis,reference tissue modeling or spectral analysis) may be applied todetermine the cutoffs. In addition, the scan data can be qualitativelyinterpreted from images that reflect the regional brain distribution ofeither SUV, Logan DVR or other parameters in which one having ordinaryskill in the art of interpreting PET scans can determine that thequalitative amount and distribution of amyloid is consistent with aprodromal phase of a clinically diagnosed amyloid deposition disease.

In another embodiment of the invention, in vivo or in vitro detection iseffected, in relation to a subject who has or who is at risk of havingat least one amyloid deposit (i.e., a deposit comprised of at least oneamyloidogenic protein), via a methodology that entails:

(a) administering to a subject suffering from a disease associated withamyloidosis, a detectable quantity of a pharmaceutical compositioncomprising at least one compound of formula (I) as described above andpharmaceutically acceptable salts thereof; and

(b) detecting the binding of the compound to an amyloid depositcomprising at least one amyloidogenic protein, wherein the amyloidogenicprotein is selected from the group consisting of AL, AH, ATTR, Aβ2M, AA,AApoAI, AApoAII, AGel, ALys, AFib, ACys, ABri, ADan, APrP, ACa1, A1APP,AANF, APro, AIns, AMed, AKer, A(tbn), and ALac.

In primary systemic amyloidosis (AL), the amyloidogenic protein can beabnormally conformed monoclonal immunoglobulin light chains (k or λ)produced by clonal plasma cells. Fibrils deposit in kidneys, heart,liver, and other organs/tissues.

In a few cases, immunoglobulin chain amyloidosis fibrils contain onlyheavy chain sequences rather than light chain sequences. In thatcircumstance, the disease is termed “heavy chain amyloidosis” (AH).

In transthyretin amyloidosis, the precursor protein is the normal ormutant sequence TTR, a transport protein synthesized in the liver andchoroid plexus. TTR is a tetramer of 4 identical subunits of 127 aminoacids each. Normal-sequence TTR forms amyloid deposits in the cardiacventricles of elderly (>70 year-old) individuals; this disease is alsocalled “senile cardiac amyloidosis.” The prevalence of TTR cardiacamyloidosis increases progressively with age, affecting 25% or more ofthe population older than 90 years. Normal-sequence ATTR can be anincidental autopsy finding, or it can cause clinical symptoms (e.g.,heart failure and arrhythmias).

Point mutations in TTR increase the tendency of TTR to form amyloid.Amyloidogenic TTR mutations are inherited as an autosomal dominantdisease with variable penetrance. More than 60 amyloidogenic TTRmutations are known. The most prevalent TTR mutations are TTR Val30Met(common in Portugal, Japan, and Sweden), and TTR Val122Ile (carried by3.9% of African Americans). Amyloidogenic TTR mutations cause depositsprimarily in the peripheral nerves, heart, gastrointestinal tract, andvitreous.

In β-microglobulin amyloidosis, the precursor protein is a normalβ-microglobulin (β2M), which is the light chain component of the majorhistocompatibility complex. In the clinical setting, Aβ2M is associatedwith patients on dialysis and, rarely, patients with renal failure whoare not on dialysis.

β2M is normally catabolized in the kidney. In patients with renalfailure, the protein accumulates in the serum. Conventional dialysismembranes do not remove β2M; therefore, serum levels can reach as highas 30-60 times the reference range values in patients on hemodialysis.Typical organs involved include the carpal ligament and, possibly, thesynovial membranes (leading to arthropathies and bone cysts) and theheart, gastrointestinal tract, liver, lungs, prostate, adrenals, andtongue.

Amyloid A (AA) amyloidosis is the most common form of systemicamyloidosis worldwide. It occurs in the course of a chronic inflammatorydisease of either infectious or noninfectious etiology. In AA, thekidney, liver, and spleen are the major sites of involvement.

Apolipoprotein AI amyloidosis (AApoAI) is an autosomal dominantamyloidosis caused by point mutations in the apoAI gene. Usually, thisamyloidosis is a prominent renal amyloid. Some kindreds have peripheralneuropathy or cardiac disease. ApoAI (likely of normal sequence) also isthe fibril precursor in localized amyloid plaques in the aortae ofelderly people.

Apolipoprotein AII amyloidosis (AApoAII) is an autosomal dominantamyloidosis caused by point mutations in the apoAII gene. The 2 kindredsdescribed with this disorder have each carried a point mutation in thestop codon, leading to production of an abnormally long protein.

The precursor protein in gelsolin amyloidosis (AGel) is theactin-modulating protein gelsolin. Amyloid fibrils include a gelsolinfragment that contains a point mutation.

Fibrinogen amyloidosis (AFib) is an autosomal dominant amyloidosiscaused by point mutations in the fibrinogen alpha chain gene.

Lysozyme amyloidosis (ALys) is an autosomal dominant amyloidosis causedby point mutations in the lysozyme gene.

The precursor protein in cystatin C amyloidosis (ACys) is cystatin C,which is a cysteine protease inhibitor that contains a point mutation.This condition is clinically termed HCHWA, Icelandic type. ACys isautosomal dominant. Clinical presentation includes multiple strokes andmental status changes beginning in the second or third decade of life.The pathogenesis is one of mutant cystatin that is widely distributed intissues, but fibrils form only in the cerebral vessels; therefore, localconditions are believed to play a role in fibril formation.

The precursor protein in prion protein amyloidosis (APrP) is a prionprotein, which is a plasma membrane glycoprotein. The etiology is eitherinfectious (i.e., kuru) or genetic (i.e., Creutzfeldt-Jakob disease(CJD), Gerstmann-Sträussler-Scheinker (GSS) syndrome, fatal familialinsomnia (FFI)). The infectious unit is the prion protein, which inducesa conformational change in a homologous protein encoded by a hostchromosomal gene. Patients with CJD, GSS, and FFI carry autosomaldominant amyloidogenic mutations in the prion protein gene; therefore,the amyloidosis forms even in the absence of an infectious trigger.

In calcitonin amyloid (ACal), the precursor protein is calcitonin, acalcium regulatory hormone synthesized by the thyroid. Patients withmedullary carcinoma of the thyroid may develop localized amyloiddeposition in the tumors, consisting of normal-sequence procalcitonin(ACal). The presumed pathogenesis is increased local calcitoninproduction, leading to a sufficiently high local concentration of thepeptide causing polymerization and fibril formation.

In islet amyloid polypeptide amyloidosis (AIAPP), the precursor proteinis an islet amyloid polypeptide (IAPP), also known as amylin. IAPP is aprotein secreted by the islet beta cells that are stored with insulin inthe secretory granules and released in concert with insulin. Normally,IAPP modulates insulin activity in skeletal muscle. IAPP amyloid isfound in insulinomas and in the pancreas of many patients with diabetesmellitus type 2.

Atrial natriuretic factor amyloidosis is associated with the precursorprotein, atrial natriuretic factor (ANF), a hormone controlling salt andwater homeostasis, which is synthesized by the cardiac atria. Amyloiddeposits are localized to the cardiac atria. This condition is highlyprevalent in elderly people. Atrial natriuretic factor amyloidosis(AANF) is most common in patients with long-standing congestive heartfailure, presumably because of persistent ANF production.

In prolactin amyloid (APro), prolactin or prolactin fragments are foundin the pituitary amyloid. This condition is often observed in elderlypeople and has also been reported in an amyloidoma in a patient with aprolactin-producing pituitary tumor.

Amyloids of the skin react with some antikeratin antibodies to generatea localized faun of amyloidosis. However, the exact identity of thefibrils is not chemically confirmed in keratin amyloid, but they arereferred to as keratin amyloid proteins (AKer).

Aortic medial amyloid occurs in most people older than 60 years. Medinamyloid (AMed) is derived from a proteolytic fragment of lactadherin, aglycoprotein expressed by mammary epithelium.

Familial British dementia (FBD) is characterized neuropathologically bydeposition of a unique amyloid-forming protein, ABri. It is a fragmentof an abnormal form of a precursor protein, BRI.

In Familial Danish dementia (FDD), a decamer duplication between codons265 and 266 in the 3′ region of the BRI gene originates an amyloidpeptide named ADan, 11 residues longer than the wild-type peptideproduced from the normal BRI gene. ADan deposits have been found widelydistributed in the CNS of FDD cases. The deposits of ADan arepredominantly non-fibrillar aggregates.

The ABri and ADan peptides are fragments derived from a larger,membrane-anchored precursor protein, termed BRI precursor protein, andencoded by the BRI gene on chromosome 13.

Pindborg tumor is characterized by the production of large amounts ofamyloid and the presence of calcified lamellar bodies. The amyloidprotein associated with this syndrome has yet to be named but iscommonly referred to as A(tbn).

Amyloid fibrils can be formed in the absence of serum amyloid P (SAP)component and heparin sulfate proteoglycans from several naturalpolypeptides, such as insulin. This gives rise to the amyloid protein,AIns, the precuror of which is insulin.

Another protein, lactoferrin, is reported as the major fibril protein infamilial subepithelial corneal amyloidosis. It is presumed that either astructural abnormality or abnormally increased concentration in theserum gives rise to the amyloid protein ALac.

The amyloidogenic proteins are detected by the present thioflavincompounds. The thioflavin compounds target at least one amyloidogenicprotein which is derived from at least one protein precursor selectedfrom the group consisting of immunoglobulin light chain, immunoglobulinheavy chain, transthyretin, (β2-microglobulin, (Apo)serum AA,Apolipoprotien AI, Apolipoprotein AII, gelsolin, lysozyme, fibrinogenα-chain, cystatin C, ABriPP, ADanPP, prion protein, (Pro)calcitonin,islet amyloid polypeptide, atrial natriuretic factor, prolactin,insulin, lactadherin, kerato-epithelin, Pindborg tumor associatedprecursor protein (tbn) and lactoferrin. It is these protein targetswhich give rise to different syndromes or diseases of affected tissues.See Buxbaum, Curr. Opin Rheumatol 16: 67-75 (2003). See also, Merliniand Westermark, J Intern Med 255: 159-178 (2004).

The following examples are given to illustrate the present invention. Itshould be understood, however, that the invention is not to be limitedto the specific conditions or details described in these examples.

EXAMPLES Synthesis Examples Example 1 Preparation of5-Methoxy-2-(4′-flurophenyl)benzofuran

2-Benzyloxy-5-methoxybenzaldehyde (I):

To a solution of 2-hydroxy-5-methoxybenzaldehyde (0.152 g, 1.0 mmol) indry MeCN (50 mL) was added 4-fluorobenzyl bromide (0.145 mg, 1.0 mmol),and anhydrous K₂CO₃ (0.27 g, 2.0 mmol). The mixture was refluxed understirring for 4 h, and then evaporated in vacuo. Water (100 mL) was addedand the mixture was extracted with EtOAc. The separated organic layerwas dried with MgSO₄, and then evaporated in vacuum. Finally, theresidue was eluted through a silica gel column with n-hexane/EtOAc (4:1)to give white compound I (0.21 g, 88%).

¹H NMR (300 MHz, acetone-d₆): δ 10.56 (s,1H, CHO), 7.35-7.48 (m, 2H),7.12-7.19 (m, 5H), 5.20 (s, 2H, OCH₂), 3.90 (s, 3H, OCH₃).

5-Methoxy-2-(4′-fluorophenyl)benzofuran (II):

To a solution of compound I (50 mg, 0.2 mmol) in dry toluene (5 mL) wasadded phosphazene base(P₄-t-Bu) solution in hexane (0.2 mL, 1.0 M), andrefluxed overnight. CHCl₂ (50 mL) was added and the solution was washedwith water. The separated organic layer was dried with MgSO₄, and thenevaporated in vacuum. Finally the residue was separated through a silicagel TCL with hexane/EtOAc(4:1) to obtain white compound II (39 g, 85%).

¹H NMR (300 MHz, acetone-d₆): δ 7.92-7.98 (m, 2H), 7.46 (d, J=8.4 Hz,1H), 7.21-7.28 (m, 3H), 7.15 (d, J=2.7 Hz, 1H), 6.90 (dd, J=3 Hz, 1H),3.90 (s, 3H, OCH₃).

5-Hydroxy-1-(4′-fluorophenyl)benzofuran (III):

A solution of compound II (24 mg, 0.1 mmol) in 5 mL methylene dichloridewas added to 2 mL of a 1.0 M solution of BBr₃ in methylene dichlorideand stirred at room temperature for 3 h. The solvent was removed byevaporation, and the crude product was purified on silica gel TLC withhexane/EtOAc (3:1) to give white compound III (21 mg, 89%).

¹H NMR (300 MHz, acetone-d₆): δ 8.15 (s, 1H), 7.92-7.95 (m, 2H), 7.37(d, J=8.7 Hz, 1H), 7.26 (t, J₁=J₂=8.9 Hz 2H), 7.14 (s, 1H), 7.02 (d,J=2.4 Hz, 1H), 6.85 (dd, J=2.4 Hz, 1H).

Example 2 Preparation of 5-Methoxy-2-(4′-nitrophenyl)benzofuran

3-Methoxy-2-(4′-nitrobenzyloxy)benzaldehyde (IV):

Compound IV was synthesized from 4-nitrophenzyl chloride as sameprocedure as preparation of compound I in 91% yield.

¹H NMR (300 MHz, acetone-d₆): δ 8.36 (d, J=9.0 Hz, 2H), 8.18 (d, J=9.0Hz, 2H), 7.26-7.29 (m, 1H), 7.21-7.26 (m, 1H), 7.01 (dd, J₁=2.4 Hz,J₂=8.4 Hz, 1H), 5.46 (s, 2H), 3.84 (s, 3H).

5-Methoxy-2-(4′-nitrophenyl)benzofuran (V):

Compound V was synthesized from compound (IV) as same procedure forCompound II in 89% yield.

¹H NMR (300 MHz, acetone-d₆): δ 8.35 (d, J=8.4 Hz, 2H), 8.15 (d, J=8.4Hz, 2H), 7.53 (d, J=8.4 Hz, 1H), 7.56 (s, 1H), 7.21 (d, J=2.3 Hz, 1H),7.01(dd, J₁=8.4 Hz, J₂=2.4 Hz, 1H), 3.84 (s, 3H).

Examples 3-6

Using the procedures analogous to those described in Examples 1 and 2,the following compounds were synthesized:

Example 3

Example 4

Example 5

Example 6

Biological Examples Mouse Brain Entry Studies

5-Methoxy-2-(4′-flurophenyl)benzofuran in ethanol was diluted withnormal saline to make a solution containing about 500 microCuries permL. Wild-type Swiss Webster mice were weighed, injected withapproximately 30 microCuries via lateral tail vein, and killed atdifferent times after injection. A whole blood sample was taken viacardiac puncture at the time of death, and the brain was rapidly removedand dissected into cerebellum and whole brain (no brain stem). A femurwas removed from each mouse as well to determine the extent ofmetabolism to [¹⁸F]fluoride. These fractions were assayed in a gammawell counter along with a calibrated portion of the injectate, andsamples were decay-corrected to the time of injection. The samples wereWeighed, and the percent injected dose per gram tissue (% ID/g) wasdetermined and normalized to mouse body weight ((% ID*kg)/g).

Baboon Brain Entry Studies

A 40 kg baboon was anesthesized, immobilized, and placed on aventilator. The baboon was positioned in a PET scanner to image thebrain, and a transmission scan was performed to correct for attenuation.The baboon was injected intravenously with a solution containing 8 mCiof 5-Methoxy-2-(4′-fluorophenyl)benzofuran, and the brain was imaged ina series of increasing data collection time bins over 2 h postinjection. Following data acquisition, the images were reconstructed andregions of interest were drawn. Decay and attention correctedtime-activity curves (TAC) were generated for each of the regions todetermine the quantitative time course of radioactivity in each brainregion. These TAC's indicate excellent brain penetration of theradiotracer and rapid clearance of radioactivity from normal baboonbrain. These in vivo properties combined with the relatively highaffinity of the ligand in vitro for aggregated amyloid indicate that thecompound is a potentially useful amyloid imaging agent.

Characterization of Binding Affinity to Aβ Synthetic Peptide

The characteristics of benzofuran derivative binding were analyzed usingsynthetic Aβ(1-40) and 2-(4′-[³H]methylamino-phenyl)-benzothiazole([³H]BTA-1) in phosphate-buffered saline (pH 7.4), as previouslydescribed. Klunk et al., Life Sci. 69:1471 (2001); Mathis et al.,Bioorg. Med. Chem. Lett., 12:295 (2002). The table below lists theinhibition constants (Ki) of the four compounds shown for competition of[³H]BTA-1 binding from synthetic Aβ(1-40). Compounds with Ki valuesbelow 20 nM are exemplary for use as in vivo PET radiotracers.

Ki Benzofurans (nM)

3.7

27.7

3.9

96

1.2

28

2.8

2

1. An amyloid binding compound of Formula (I) or a pharmaceuticallyacceptable salt thereof:

wherein Y is H, NO₂, —NR″₃ ⁺, F, Cl, Br, I, O—(CR″₂)_(n)—X, or—(CR″₂)_(n)—X; wherein X is F, Cl, Br or I; and n is 1-5; R′ is H or alower alkyl group; R″ is H or a lower alkyl group; R₃-R₁₀ areindependently selected from the group consisting of H, F, Cl, Br, I,C₁-C₅ alkyl, (CH₂)₁₋₃—OR₁₁, CF₃, —(CH₂)₁₋₃—X, —O—(CH₂)₁₋₃—X, CN,—CO—R₁₁, —N(R₁₁)₂, —NR″₃ ⁺, —NO₂, —CO—N(R₁₁)₂, —O—(CO)—R₁₁, OR₁₁, SR₁₁,COOR₁₁, R_(ph), —CR₁₁═CR₁₁—R_(ph) and —C(R₁₁)₂—C(R₁₁)₂—R_(ph), wherein Xis F, Cl, Br or I; and R_(ph) is phenyl optionally substituted with oneor more substituents selected from the group consisting of F, Cl, Br, I,C₁-C₅ alkyl, (CH₂)₁₋₃—OR₁₁, CF₃, —(CH₂)₁₋₃—X, —O—(CH₂)₁₋₃—X, CN,—CO—R₁₁, —N(R₁₁)₂, —CO—N(R₁₁)₂, —O—(CO)—R₁₁, OR₁₁, SR₁₁, and COOR₁₁,wherein each R₁₁ is independently H or C₁-C₅ alkyl.
 2. The amyloidbinding compound according to claim 1, wherein Y is H, NO₂, —NR″₃ ⁺, F,Cl, Br, I, or —(CR″₂)_(n)—X.
 3. The amyloid binding compound accordingto claim 2, wherein R₃-R₁₀ are independently selected from the groupconsisting of H, F, Cl, Br, I, —N(R₁₁)₂, and OR₁₁.
 4. The amyloidbinding compound according to claim 3, wherein R⁸ and R⁹ areindependently OR₁₁.
 5. The amyloid binding compound according to claim4, wherein each of R₇ and R₁₀ is H.
 6. The amyloid binding compoundaccording to claim 5, wheren each of R₃, R₄, R₅, and R₆ is H.
 7. Theamyloid binding compound according to claim 2, wheren Y is selected fromF, Cl, Br, I, and —NO₂.
 8. The amyloid binding compound according toclaim 7, wherein Y is F.
 9. The amyloid binding compound according toclaim 2, wherein each of R₃, R₄, R₅, and R₆, R₇, and R₁₀ is H, and R₈and R₉ are independently OR₁₁.
 10. The amyloid binding compoundaccording to claim 2, wherein Y comprises the at least one thedetectable label.
 11. (canceled)
 12. (canceled)
 13. The amyloid bindingcompound according to claim 1 having the following formula:


14. A pharmaceutical composition comprising (i) an effective amount ofan amyloid binding compound according to claim 1; and (ii) apharmaceutically acceptable carrier. 15.-33. (canceled)
 34. A method ofselectively binding an amyloid binding compound of claim 1 or apharmaceutically acceptable salt thereof to amyloid plaques but notneurofibrillary tangles in brain tissue which contains both, the methodcomprising contacting the amyloid plaques in in vitro binding orstaining assays with a compound of claim 1 at a concentration belowabout 10 nM.
 35. A method of selectively binding in vivo an amyloidbinding compound of Formula (I) or a pharmaceutically acceptable saltthereof to amyloid plaques but not to neurofibrillary tangles in braintissue which contains both, the method comprising administering aneffective amount of a compound of claim 1 or a pharmaceuticallyacceptable salt thereof such that the blood concentration of theadministered compound remains below about 10 nM in vivo. 36.-73.(canceled)